Compositions and Methods for Treating Tumors, Fibrosis, and Pulmonary Alveolar Proteinosis

ABSTRACT

The present disclosure provides pharmaceutical compositions and methods useful for modulating angiogenesis and for inhibiting metastasis, tumors, pulmonary alveolar proteinosis, and fibrosis in a mammalian tissue. Pharmaceutical compositions and methods include inhibitors of LOXL2 expression and activity, such as shRNA targeting LOXL2.

CROSS-REFERENCE

This application claims the benefit of Israeli Patent Application No.184627, filed Jul. 15, 2007, which is incorporated herein by reference.

BACKGROUND

Lysyl oxidase (LO or LOX) is a copper containing amine oxidase thatoxidizes primary amine substrates to reactive aldehydes. LOX catalyzesoxidative deamination of peptidyl lysine and hydroxylysine residues incollagens, and peptidyl lysine residues in elastin, and aids in theformation of the extracellular matrix. The resulting peptidyl aldehydestypically condense and undergo oxidation reactions to form thelysine-derived covalent cross-links required for the normal structuralintegrity of the extracellular matrix. Hydrogen peroxide (H₂O₂) andammonium are usually released in quantities stoichiometric with thepeptidyl aldehyde product.

LOX can oxidize certain lysine residues in collagen and elastin outsideof the cell; however, it may also act intracellularly, where it mayregulate gene expression. In addition, LOX can induce chemotaxis ofmonocytes, fibroblasts and smooth muscle cells. LOX itself can beinduced by a number of growth factors and steroids such as TGF-β, TNF-αand interferon (Csiszar, Prog. Nucl. Acid Res. 70:1-32 (2001)). LOX hasalso been implicated in diverse biological functions such asdevelopmental regulation, tumor suppression, cell motility, and cellularsenescence. The diverse role of LOX and its recently discovered aminooxidase family members, lysyl oxidase related or lysyl oxidase-likeproteins (LOR or LOXL), may play important roles with respect to theirintracellular and extracellular localization.

The expression or implication of LOX and LOXL in diseases may also vary.This may be due to a number of reasons, such as the difference in tissuedistribution, processing, domains, regulation of activity, as well asother differences between the proteins. For example, LOX and LOXL areimplicated in fibrotic diseases as both LOX and LOXL are highlyexpressed in myo-fibroblasts around fibrotic areas (Kagen, Pathol. Res.Pract. 190:910-919 (1994); Murawaki et al., Hepatology 14:1167-1173(1991); Siegel et al., Proc. Natl. Acad. Sci. USA 75: 2945-2949 (1978);Jourdan Le-Saux et al., Biochem. Biophys. Res. Comm. 199:587-592 (1994);Kim et al., J. Cell Biochem. 72:181-188 (1999)). LOX and the variousLOXL are also implicated in a number of cancers. For example, LOXL andLOXL4 have been shown to be epigenetically silenced and can inhibitras/extracellular signal-regulated kinase signaling pathway in humanbladder cancer (Wu et al., Cancer Res. 67:4123-4129 (2007)). Others haveshown selective upregulation and amplification of the LOXL4 gene in headand neck squamous cell carcinoma (Gorough et al., J. Pathol. 212:74-82(2007)). LOX and LOXL2 have also been implicated in a number of tumors,such as colon and esophageal cancers (Csiszar, Prog. Nucl. Acid Res.70:1-32 (2001)). In breast cancer, LOX and the LOXL family members havebeen linked to cancer (Kirschmann et al., Cancer Res. 62:448-4483(2002)).

Thus, there is a need for compositions and methods to modulate LOX andLOXL activity. One such method is through the use of RNA interference(RNAi). RNAi refers to methods of sequence-specific post-transcriptionalgene silencing which is mediated by a double-stranded RNA (dsRNA) calleda short interfering RNA (siRNA). RNAi is an endogenous mechanism thatuses small noncoding RNAs to silence gene expression. When an siRNA isintroduced into a cell, it binds to the endogenous RNAi machinery toalter the level of mRNA containing complementary sequences with highspecificity. The RNAi response involves an endonuclease complex known asthe RNA-induced silencing complex (RISC), which mediates cleavage of asingle-stranded RNA complementary to the antisense strand of the siRNAduplex. Cleavage of the target RNA takes place in the middle of theregion complementary to the antisense strand of the siRNA duplex(Elbashir et al., Genes Dev. 15:188-200, (2001)).

As a result, there is a need for compositions to modulate LOX and LOXL,such as through the use of RNAi. Methods for using such compositions totreat and diagnose conditions are also needed. The present disclosureaddresses these needs and provides other advantages as well.

SUMMARY

The present disclosure provides pharmaceutical compositions and methodsuseful for modulating angiogenesis and fibrosis, and for treatingcancer, by inhibiting metastasis and tumors in a subject, such asprimary tumors. Moreover, the expression of LOXL2 is correlated withpulmonary alveolar proteinosis and as such can be used for accuratediagnosis and treatment of pulmonary alveolar proteinosis (PAP).

In one aspect, the present disclosure provides an isolatedpolynucleotide comprising a first sequence hybridizable to apolynucleotide sequence encoding LOXL2 or SEQ ID NO. 2, a secondsequence complementary to the first sequence, and a linking sequencethat joins the first sequence to the second sequence. The linkingsequence can form a hairpin loop structure. The first sequence cancomprise SEQ ID NO. 20 or 21. Also provided is an isolatedpolynucleotide comprising SEQ ID NO. 20 or 21. The isolatedpolynucleotide can be at least twice the length of SEQ ID NO. 20 or 21.The isolated polynucleotide comprising SEQ ID NO. 20 may furthercomprise a second sequence complementary to it. Alternatively, theisolated polynucleotide comprising SEQ ID NO. 21 may further comprise asecond sequence complementary to it. The isolated polynucleotidescomprising SEQ ID NO. 20 or 21 may also comprise a hairpin loopstructure. Further provided are expression vectors comprising theisolated polynucleotides as well as host cells comprising the expressionvectors.

In another aspect, pharmaceutical compositions comprising apolynucleotide comprising a first sequence hybridizable to apolynucleotide sequence encoding LOXL2 or SEQ ID NO. 2, a secondsequence complementary to the first sequence, and a linking sequencethat joins the first sequence to the second sequence; and, apharmaceutical excipient, are provided. Also provided are pharmaceuticalcompositions comprising a polynucleotide comprising SEQ ID NO. 20 or 21;and, a pharmaceutical excipient.

The present disclosure also provides methods of administering thecompositions described herein. Methods for inhibiting primary tumorgrowth, metastasis, fibrosis, or angiogenesis in a subject are alsodisclosed. The methods can comprise administering to the subject aneffective amount of a polynucleotide to inhibit primary tumor growth,metastasis, fibrosis, or angiogenesis in the subject. The polynucleotidecan comprise a first sequence hybridizable to a polynucleotide sequenceencoding LOXL2 or SEQ ID NO. 2, a second sequence complementary to thefirst sequence, and a linking sequence that joins the first sequence tothe second sequence. The polynucleotide can comprise a linking sequencethat forms a hairpin loop structure. The first sequence can comprise SEQID NO. 20 or 21. The methods can also encompass administering to asubject a polynucleotide comprising SEQ ID NO. 20 or 21 to inhibitprimary tumor growth, metastasis, fibrosis, or angiogenesis in thesubject.

Methods for treating PAP in a subject are also provided. The presentdisclosure provides methods comprising administering to the subject aneffective amount of an agent to inhibit PAP, wherein the agent modulatesthe expression or activity of a lysyl oxidase or lysyl oxidase likeprotein. Methods for detecting PAP in a subject are also provided,wherein the subject is administered an agent that detects the expressionor activity of a lysyl oxidase or lysyl oxidase like protein, whereinthe expression or activity is used to diagnose PAP in the subject. Thelysyl oxidase level or activity may be that of LOXL2, LOXL3, or both,and the agent used to treat or diagnose PAP can be an antibody, a smallmolecule, antisense molecule, ribozyme, DNAzyme, triple helix formingoligonucleotides, siRNA, or shRNA. The agent may be an inhibitor oflysyl oxidase or lysyl oxidase like protein, such as LOXL2, LOXL3, orboth.

INCORPORATION BY REFERENCE

All publications, patents, patent applications and sequences identifiedby their accession numbers mentioned in this specification are hereinincorporated in their entirety by reference into the specification, tothe same extent as if each individual publication, patent, patentapplication or sequence identified by their accession number wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentinvention may be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawingsof which:

FIG. 1 illustrates LOXL2 shRNA sequences. Each sequence forms a hairpinas the underlined sequences in each are sense/antisense strands. TheshRNA are expressed using the pLKO.1-puro vector (available from Sigma)to allow for transient or stable transfection of the shRNA as well asproduction of lentiviral particles.

FIG. 2 is a schematic illustrating the tumor invasion assay.

FIG. 3 illustrates the expression of LOXL2 in (A) MDA-MDB 231/LM2-4 andHT1080 cancer cells and in (B) MDA-MB 231 breast cancer and YU/PAC2melanoma cells, infected with lentiviral vectors directing expression of195 or 197 LOXL2 specific shRNA (also referred to as sh.Loxl2.195 orsh.Loxl2.197, respectively)

FIG. 4 is a microphotograph illustrating the morphological shift incells infected with lentiviral vectors directing expression ofsh.Loxl2.195 or sh.Loxl2.197.

FIG. 5 is (A) a microphotograph illustrating the effect of lentiviralvectors directing expression of sh.Loxl2.195 or sh.Loxl2.197 onMDA-MB-231 breast cancer cells and HT1080 fibrosarcoma cells in thetumor invasion assay. (B) is a Western blot showing the knockdown ofLOXL2 expression and (C) show the number of invading cells per field.

FIG. 6 is a photomicrograph illustrating the expression of LOXL2 andLOXL3 in normal and PAP lung tissue.

FIG. 7 illustrates rhodamine phalloidin staining of MDA-231 cells. (A)Wild type MDA-231 cells and control-infected lentiviral stable MDA-231cells with phalloidin staining of F-actin reveal long fibrils typical ofa cell that has undergone epithelial-mesenchymal transition (EMT). (B)MDA-231 cells infected with sh.Loxl2.195 are depleted for LOXL2 andrevealed a “rim” effect near the cell membrane, more typical of a normalepithelial cell.

FIG. 8 illustrates primary tumor development in mice injected withMDA-231 si-control (si-c) or sh.Loxl2.195 (si-195) cells. MDA-231 cellswere infected with control shRNA encoding lentivirus or lentivirusvector expressing sh.Loxl2.195. The cells were selected with puromycinand Loxl2 expression determined prior to injection into the mammary fatpads of balb/c nu/nu female mice. (A) Tumor volume was measured 6, 11,14, 18, 22, 25, and 27 days after injection. (B) illustrates tumorweight from MDA-231 si-cont vs. sh.Loxl2.195 (si-195) mice 27 days afterinjection.

FIG. 9 illustrates genes in cancer cells affected by LOXL2overexpression or inhibition of Loxl2 expression. (A) Genes upregulatedin MCF-7 cells overexpressing recombinant LOXL2. Gene expression wasmeasured by RT-PCR and Western blotting. Cells were transfected with atetracycline-regulated construct expressing LOXL2 (clones 12 and 14),control lentiviral vector containing a non-related shRNA (WT), or amutant of LOXL2 that is enzymatically inactive due to a mutation in itsLTQ motif (Y689F). (B) Infection with lentivirus directed expression ofshRNA directed against Loxl2. C=control shRNA, L2=sh.Loxl2.195

FIG. 10 illustrates expression of LOXL2 is enhanced by hypoxia. (A)Cells were incubated in a hypoxia chamber. Control was incubated innormoxic conditions (regular incubator). RNA was prepared from the cellsat the end of the experiment and amplified by RT-PCR. PC=cellsexpressing recombinant LOXL2, NC=PCR without RT. (B) LOXL2 levels wereassessed by Western blot. Cells were stimulated for the indicated timeswith the indicated concentration of CoCl₂. Equal concentrations of celllysates were prepared with lysis buffer. Cell lysates were separated ona gradient SDS/PAGE gel, blotted and probed with our anti-LOXL2antibodies. Membranes were stripped and reprobed with an antibodydirected against β-actin to verify equal loading.

FIG. 11 illustrates expression of a cell surface bound LOXL2 receptor inhuman umbilical vein derived endothelial cells (HUVEC). (A) LOXL2 wasiodinated and four different cell lines were tested for their specificbinding to LOXL2. Iodinated LOXL2 was added to each well and competitionwas done with unlabeled LOXL2. (B) LOXL2 does not bind specifically togelatin, laminin or fibronectin. Iodinated LOXL2 was added to each welland competition was done with unlabeled LOXL2. There was no specificbinding demonstrating the binding observed to the cells is not caused bybinding to the ECM components fibronectin, laminin, or gelatin. (C)Iodinated LOXL2 was added to each well and competition was done withunlabeled LOXL2. In the absence or addition of 100 ug/ml heparin(heparin does not inhibit the binding) or after prior digestion withheparinase (does not affect the binding to the putative receptor).

FIG. 12 illustrates expression of LOXL2 and LOXL3 in neuronal cells ofthe central nervous system (CNS). In-situ hybridization on tissuesections from normal human brain cortex was performed using probesdirected at LOX, LOXL1, LOXL2, or LOXL3. S=sense, as=antisense.

DETAILED DESCRIPTION

The principles and operation of the present disclosure may be betterunderstood with reference to the drawings and accompanying description.It is to be understood that disclosure is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawingsdescribed in the Examples section. The disclosure is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

The present disclosure provides pharmaceutical compositions and methodsthat can be used to modulate angiogenesis and to inhibit tumor growth,tumor invasiveness and tumor fibrosis. For example, the presentdisclosure can be used to suppress tumor growth and metastasis as wellas to treat and diagnose disorders such as, for example, arthritis,diabetic retinopathy, psoriasis, vasculitis and PAP.

The innovative methods and compositions described include the use of aninhibitor of LOX or LOXL, such as agents that inhibit LOXL2. An exampleof LOX or LOXL include the enzyme having an amino acid sequencesubstantially identical to a polypeptide expressed or translated fromone of the following sequences: EMBL/GenBank accessions: M94054;AAA59525.1—mRNA; 545875; AAB23549.1—mRNA; 578694; AAB21243.1—mRNA;AF039291; AAD02130.1—mRNA; BC074820; AAH74820.1—mRNA; BC074872;AAH74872.1—mRNA; M84150; AAA59541.1—Genomic DNA. Particular examples ofLOXL are described in Molnar et al., Biochim Biophys Acta. 1647: 220-24(2003); Csiszar, Prog. Nucl. Acid Res. 70:1-32 (2001); and in WO01/83702. (It is noted that in these 3 publications, “LOXL1” wasreferred to as “LOXL” whereas in the present invention “LOXL” isreferred to a lysyl oxidase-like protein in general, not just LOXL1.)These enzymes include LOXL1, encoded by mRNA deposited at GenBank/EMBLBC015090; AAH15090.1; LOXL2, encoded by mRNA deposited at GenBank/EMBLU89942; LOXL3, encoded by mRNA deposited at GenBank/EMBL AF282619;AAK51671.1; and LOXL4, encoded by mRNA deposited at GenBank/EMBLAF338441; AAK71934.1.

LOX or LOXL also encompasses a functional fragment or a derivative thatstill substantially retains its enzymatic activity catalyzing thedeamination of lysyl residues. Typically, a functional fragment orderivative retains at least 50% of its lysyl oxidase activity. Afunctional fragment or derivative can retain at least 60%, 70%, 80%,90%, 95%, 99% or 100% of its lysyl oxidase activity. A LOX or LOXL caninclude conservative amino acid substitutions that do not substantiallyalter its activity. Suitable conservative substitutions of amino acidsare known to those of skill in this art and may be made generallywithout altering the biological activity of the resulting molecule.Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity.

Inhibitors used may include inhibitors of the lysyl oxidase family ofenzymes which catalyze the formation of covalent crosslinks betweenlysine residues on adjacent collagen or elastin fibrils. At least fivedifferent lysyl oxidases are known to exist in both humans and mice, LOXand four LOX related, or LOX-like proteins: LOXL1 (or LOL), LOXL2 (orLOR-1), LOXL3 (or LOR-2), and LOXL4 (or Lox-C). The five forms of lysyloxidases reside on five different chromosomes. These family members showsome overlap in structure and function, but appear to have distinctfunctions as well. For example, LOX appears to be lethal at parturitionin mice (Hornstra et al., J. Biol. Chem. 278:14387-14393 (2003)),whereas LOXL deficiency causes no severe developmental phenotype(Bronson et al., Neurosci. Lett. 390:118-122 (2005)). The lysyl oxidasefamily includes four genes, such as those with SEQ ID NOs. 1, 4, 5, or7, or enzymes with the amino acid sequences in SEQ ID NOs: 2, 3, 6, 8,or 9.

LOX has highly conserved protein domains, conserved in several speciesincluding human, mouse, rat, chicken, fish and Drosophila. The human LOXfamily has a highly conserved C-terminal region containing the 205 aminoacid LOX catalytic domain. The conserved region contains the copperbinding (Cu), cytokine receptor like domain (CRL), and thelysyl-tyrosylquinone cofactor site (LTQ). Twelve cysteine residues arealso similarly conserved, two being present in the prepropeptide regionand ten in the catalytically active processed form of LOX (Csiszar,Prog. Nucl. Acid Res. 70:1-32 (2001)).

The prepropeptide region of LOX contains a signal peptide that iscleaved. The cleavage site is predicted to be between Cys21-Ala22,generating a 16 (or 21) signal sequence and a 48 kDa amino acidpropeptide form of LOX, which is, without being bound by theory, stillinactive. Without being limited by theory, the propeptide isN-glycosylated during passage through the Golgi yielding a 50 kDainactive proenzyme that is secreted into the extracellular environmentwhere the proenzyme, or propeptide, is cleaved between Gly168-Asp169 bya metalloendoprotease, a procollagen C-proteinase, which are products ofthe Bmp1, Tll1 and Tll2 genes. BMP I (bone morphogenetic protein I) is aprocollagen C-proteinase that processes the propeptide to yield afunctional 30 kDa enzyme and an 18 kDa propeptide. The sequence codingfor the propeptide is typically moderately (approximately 60-70%)conserved, whereas the sequence coding for the C-terminal 30 kDa regionof the proenzyme in which the active site is located is usually highlyconserved (approximately 95%). (Kagan and Li, J. Cell. Biochem.88:660-672 (2003); Kagan et al., J. Cell Biochem. 59:329-38 (1995)). TheN-glycosyl units are usually subsequently removed.

Similar potential signal peptides have been predicted at the aminotermini of LOXL, LOXL2, LOXL3, and LOXL4. The predicted signal cleavagesites are between Gly25-Gln26 for LOXL, between Ala25-Gln26, for LOXL2,and between Gly25-Ser26 for LOXL3. The consensus for BMP-1 cleavage inprocollagens and pro-LOX is between Ala/Gly-Asp, and often followed byan acidic or charged residue. A potential cleavage site to generateactive LOXL is Gly303-Asp304, however, it is then followed by anatypical Pro. LOXL3 also has a potential cleavage site at Gly44y-Asp448,which is followed by an Asp, processing at this site may yield an activepeptide of similar size to active LOX. A potential cleavage site ofBMP-1 was also identified within LOXL4, at residues Ala569-Asp570 (Kimet al., J. Biol. Chem. 278:52071-52074 (2003)). LOXL2 protein may alsobe processed analogously to the other LOX family members.

A feature that may differ amongst the lysyl oxidases and lysyl oxidaselike proteins is the scavenger receptor cysteine rich (SRCR) domains.LOX and LOXL appear to lack SRCR domains, whereas LOXL2, LOXL3, andLOXL4 each have four SRCR domains at the N-terminus. SRCR domainsmediate ligand binding in a number of secreted and receptor proteins(Hoheneste et al., Nat. Struct. Biol. 6: 228-232 (1999); Sasaki et al.,EMBO J. 17:1606-1613 (1998)). Another domain that appears to be uniqueto LOXL is the presence of a proline rich domain (Molnar et al.,Biochimica Biophysica Acta 1647: 220-224 (2003)).

Tissue distribution may also differ amongst LOX and the various LOXL.For example, as shown in FIG. 12, LOXL2 and LOXL3 are highly expressedin neuronal cells, whereas LOX and LOXL1 are not. Thus, in one aspect,the present disclosure encompasses modulating expression of LOX or LOXLin the CNS, such as in the brain, or more specifically in neuronalcells.

Each member of the LOX family of enzymes includes a highly conservedlysyl oxidase domain, the activity of which is highly dependent on thepresence of copper. Removal of copper from tumor tissues leads toinhibition of angiogenesis (Rabinovitz, J. Natl. Cancer Inst.91:1689-1690 (1999); Yoshida et al., Neurosurgery 37: 287-292 (1995)).This further substantiates the role of the lysyl oxidase family ofenzymes in angiogenesis as, without being bound by theory, removal ofcopper leads to inhibition of lysyl oxidases.

Further support to the angiogenic activity of lysyl oxidases is providedby the PF4-LOXL2 binding assays. PF4 is an inhibitor of angiogenesis. Assuch, the anti-angiogenic activity exhibited by PF4 may be, withoutbeing limited by theory, effected through LOXL2 inhibition, which ishighly expressed in the endothelial cells lining blood vessels. Thusaccording to one aspect of the present disclosure, methods of modulatingangiogenesis are provided.

Angiogenesis

In an adult, formation of new blood vessels in normal or diseasedtissues is typically regulated by two processes, recapitulatedvasculogenesis (the transformation of pre-existing arterioles into smallmuscular arteries) and angiogenesis, the sprouting of existing bloodvessels (which occurs both in the embryo and in the adult). Furthermore,LOXL2 expression is induced under hypoxic conditions (FIG. 10), asangiogenesis is thought to be spurred in cancers to overcome hypoxicconditions.

The process of angiogenesis is regulated by biomechanical andbiochemical stimuli. Angiogenic factors such as vascular endothelialgrowth factor (VEGF) and basic fibroblast growth factor (bFGF) arereleased by vascular cells, macrophages, and cells surrounding bloodvessels. These angiogenic factors activate specific proteases that areinvolved in degradation of the basement membrane. As a result of thisdegradation, vascular cells migrate and proliferate thus leading to newblood vessel formation. Peri-endothelial cells, such as pericytes in thecapillaries, smooth muscle cells in larger vessels and cardiac myocytesin the heart are recruited to provide maintenance and modulatoryfunctions to the forming vessel.

The establishment and remodeling of blood vessels is controlled byparacrine signals, many of which are mediated by protein ligands whichmodulate the activity of transmembrane tyrosine kinase receptors. Amongthese molecules are vascular endothelial growth factor (VEGF) and itsreceptor families (VEGFR-1, VEGFR-2, neuropilin-1 and neuropilin-2),Angiopoietins 1-4 (Ang-1, Ang-2 etc.) and their respective receptors(Tie-1 and Tie-2), basic fibroblast growth factor (bFGF), plateletderived growth factor (PDGF), and transforming growth factor β (TGF-β).

The growth of solid tumors is limited by the availability of nutrientsand oxygen. When cells within solid tumors start to produce angiogenicfactors or when the levels of angiogenesis inhibitors decline, thebalance between anti-angiogenic and angiogenic influences is perturbed,initiating the growth of new blood vessels from the existing vascularbed into the tumor. This event in tumor progression is known as theangiogenic switch. Inhibitors of tumor angiogenesis are able to inhibittumor growth in mice, in some cases, it appears to completely inhibittumor growth and also inhibit tumor metastasis, a process that reliesupon close contact between the vasculature and tumor cells. Angiogenesisplays an important role in the progression of breast cancer.

Such findings have prompted the use of known anti-angiogenic factors inbreast cancer therapy (Klauber et al., Cancer Res. 57:81-86 (1997);Harris et al., Breast Cancer Res. Treat. 38, 97-108 (1996);Weinstatsaslow et al., Cancer Res. 54;6504-6511 (1994)). During the pastdecade several novel inhibitors of angiogenesis have been isolatedincluding inhibitors of VEGF signaling (Neufeld et al., FASEB J. 13:9-22(1999)) and inhibitors of processes which lead to the maturation andstabilization of new blood vessels. Anti-integrin antibodies have beenused as inhibitors of blood vessel maturation (Brooks et al., Cell79:1157-1164 (1994); Brooks et al., Cell 92:391-400 (1998)).

Although several anti-angiogenic drugs are now available commercially,the anti-angiogenic mechanisms of most of these drugs (e.g., angiostatinand endostatin) remain unclear (O'Reilly et al., Cell 88: 277-285(1997); O'Reilly et al., Nature Med. 2:689-692 (1996)). Sinceangiogenesis can be initiated by many (possibly compensatory) angiogenicfactors, anti-angiogenic factors which target later processes in theangiogenic response such as vessel maturation or a combination ofanti-angiogenic factors are likely to be effective in arresting vesselformation.

Platelet factor-4 (PF4) is an anti-angiogenic protein normallysequestered in platelets (Tanaka et al., Nature Med. 3:437-442 (1997);Maione et al., Science 247:77-79 (1990); Neufeld et al., The CytokineReference: A compendium of cytokines and other mediators of host defense(Oppenheim, J. J. and Feldmann, M. eds) Academic Press (2000)). PF4inhibits angiogenesis using poorly defined mechanisms (Gengrinovitch etal., J. Biol. Chem. 270:15059-15065 (1995); Brown and Parish,Biochemistry 33:13918-13927 (1994); Gupta and Singh, J. Cell Biol.127:1121-1127 (1994); Watson et al., J. Clin. Invest. 94: 261-268(1994)). It was previously speculated that PF4 binds to cell surfaceheparan-sulfate proteoglycans and in this manner inhibits the activityof angiogenic growth factors such as basic fibroblast growth factor(Watson et al., J. Clin. Invest. 94: 261-268 (1994)).

For example, the compositions and methods described in the presentdisclosure can be used to suppress tumor growth by inhibitingangiogenesis, or by directly inhibiting tumor growth (such as primarytumor growth), suppressing metastasis, as well as to treat and diagnosedisorders such as, for example, arthritis, diabetic retinopathy,psoriasis and vasculitis and primary pulmonary alveolar proteinosis.

Metastatic and Primary Tumors

The prevention, reduction, and diagnosis of tumors are important in theprevention and treatment of cancer. The transition from a localizedtumor to an invasive and metastatic tumor represents a landmark in thedevelopment of malignant disease, since it is usually associated with amarkedly worse prognosis. The understanding of the processes that governthis transition is therefore of prime importance, and LOX and LOXL rolesin these processes can be used to not only further understanding of thisprocess, but also be used to treat, prevent, or diagnosis primary andmetastatic tumors.

For example, LOXL2 expression can be decreased in breast cancer cells,melanoma cells and fibrosarcoma cells using shRNA or siRNA (FIGS. 3,5B). Administration of shRNA or siRNA targeting LOXL2 led to an EMT toMET like transition (FIG. 4, FIG. 7) as well as decrease in cellinvasion (FIG. 5), further supporting the role of LOXL2 in tumormetastasis, and by modulating LOXL2 expression can aid in inhibitingmetastatic activity. Inhibition of LOXL2 can also be used in inhibitingtumor growth and reducing primary tumors. Tumor growth inhibition can bepreventative. Alternatively, inhibition of primary tumor growth can be areduction of tumor mass, for example, tumor mass can be reduced comparedto size or volume of tumor when initially detected. The inhibition canbe in the rate of growth of the primary tumor, for example, the primarytumor mass or volume increases at a slower rate in comparison to asubject not treated with compositions disclosed herein, for example, asshown in FIG. 8.

Breast Cancer

In breast cancer, the transition from a localized to aninvasive/metastatic tumor is associated in many cases with the formationof fibrotic foci and desmoplasia, which is the presence of unusuallydense collagenous stroma, within the primary tumor (Colpaert et al., Am.J. Surg. Pathol. 25, 1557 (2001); Hasebe et al., Pathology International50: 263-272 (2000)). A similar correlation may exist in other types ofcancers such as colon and pancreatic cancers (Nishimura et al., VirchowsArch. 433:517-522 (1998); Ellenrieder et al., Int. J. Cancer 85:14-20(2000)). These observations represent apparent paradoxes at firstglance, since invasiveness has long been associated with the destructionof extracellular matrix by extracellular matrix degrading enzymes likemetalo-proteases (Stamenkovic, Semin. Cancer Biol. 10:415-433 (2000);Duffy et al., Breast Cancer Res. 2: 252-257 (2000)) and heparanase(Vlodaysky and Friedmann, J. Clin. Invest 108:341-347 (2001)). However,it is possible that deposition of excess extracellular matrix maystimulate in turn expression of matrix degrading enzymes that willcontribute under certain circumstances to tumor invasion. In fact, thereis some evidence that an increase in extracellular matrix deposition canindeed influence the production of extracellular matrix degradingenzymes (Schuppan et al., Semin. Liver Dis. 21:351-372 (2001); Swada etal., Int. J. Oncol. 19:65-70 (2001)).

Colon Cancer

Cancer of the gastrointestinal (GI) tract, especially colon cancer, is ahighly treatable and often a curable disease when localized to thebowel. Surgery is the primary treatment and results in cure inapproximately 50% of patients. Recurrence following surgery is a majorproblem and often is the ultimate cause of death. Nearly all cases ofcolorectal cancer arise from adenomatous polyps, some of which matureinto large polyps, undergo abnormal growth and development, andultimately progress into cancer. This progression would appear to takeat least 10 years in most patients, rendering it a readily treatableform of cancer if diagnosed early, when the cancer is localized.

The standard procedures currently used for establishing a definitivediagnosis for a GI tract cancer include barium studies, endoscopy,biopsy, and computed tomography (Brennan et al., Cancer: Principles andPractice of Oncology, Fourth Edition, pp. 849-882, Philadelphia, Pa.: J.B. Lippincott Co. (1993)).

The prognosis of colon cancer is typically related to the degree ofpenetration of the tumor through the bowel wall and the presence orabsence of nodal involvement. These two characteristics usually form thebasis for staging systems developed for this disease. Staging is usuallyperformed by a pathologist on tissue sections obtained via biopsy and/orsurgery and it aims to determine the anatomic extent of the disease.Accurate staging is critical for predicting patient outcome andproviding criteria for designing optimal therapy. Inaccurate staging canresult in poor therapeutic decisions and is a major clinical problem incolon cancer.

Primary Alveolar Proteinosis (PAP)

PAP is a rare lung disorder of unknown etiology characterized byalveolar filling with floccular material that stains positive using theperiodic acid-Schiff (PAS) method and is derived from surfactantphospholipids and protein components. LOXL2 and LOXL3 are likely to havea role in PAP as both are expressed in PAP tissue, but not normal lungtissue (FIG. 6)

Two forms are recognized, (1) primary (idiopathic) and (2) secondary(due to lung infections; hematologic malignancies; and inhalation ofmineral dusts such as silica, titanium oxide, aluminum, andinsecticides). Incidence of PAP is increased in patients withhematologic malignancies and AIDS, suggesting a relationship with immunedysfunction.

The alveoli in PAP are filled with proteinaceous material, which hasbeen analyzed extensively and determined to be normal surfactantcomposed of lipids and surfactant-associated proteins A, B, C, and D(SP-A, SP-C, SP-D). Evidence exists of a defect in the homeostaticmechanism of either the production of surfactant or the clearance byalveolar macrophages and the mucociliary elevator. A clear relationshiphas been demonstrated between PAP and impaired macrophage maturation.

PAP has an estimated prevalence of 1 case per 100,000 population, andmortality rates of as high as 30% within several years of disease onsethave been reported previously. The actual mortality rate may be lessthan 10%. Incidence for males is 4 times higher than for females.Patients are typically 20-50 years old at presentation.

Patients with PAP typically present with a gradual onset of symptoms. Asmany as 30% of patients are asymptomatic, even with diffuse chestradiograph (CXR) abnormalities. Symptoms can include the following:persistent dry cough (or scant sputum production), progressive dyspnea,fatigue and malaise, weight loss, intermittent low-grade fever and/ornight sweats, pleuritic chest pain, cyanosis, and hemoptysis.

The etiology of PAP is unknown. Causes may include inhalation of silicadust (acute silicoproteinosis), exposure to insecticides, aluminum dust,titanium dioxide, and other inorganic dusts, hematologic malignancies,myeloid disorders, lysinuric protein intolerance, HIV infection (AIDS),leflunomide-case report and disease-modifying antirheumatoid arthritistherapy. Differentials may include hypersensitivity pneumonitis, lungcancer, non-small cell lung cancer, oat cell lung cancer (Small Cell),Pneumocystis carinii pneumonia, pulmonary edema and cardiogenicsarcoidosis. The diagnosis can be made by lavage, if PAS staining isrequested. Therefore, PAP is probably underdiagnosed.

Lung biopsies are classically used in diagnosing for PAP: Alveoli arefilled with nonfoamy material. Transbronchial biopsies are adequate, andopen lung biopsy is not required.

Management of PAP depends on the progression of the illness, coexistinginfections, and degree of physiological impairment. The standard of carefor PAP is mechanical removal of the lipoproteinaceous material bywhole-lung lavage, which is often repeated. Historically, patients havebeen treated with systemic steroids, mucolytics (aerosol), andproteinase (aerosol) without much success. In secondary PAP, appropriatetreatment of the underlying cause also is warranted. GM-CSF has beenshown to improve PAP in several patients and is being investigated.Congenital PAP responds favorably to lung transplantation.

Lung transplantation is the treatment of choice in patients withcongenital PAP and in adult patients with end-stage interstitialfibrosis and cor pulmonale. The major complications are lung infectionswith N asteroides, Pneumocystis carinii, and/or Mycobacteriumavium-intracellulare. Pulmonary fibrosis and/or cor pulmonale also cancomplicate PAP.

Thus, to increase the accuracy of therapy and the survival rate of PAPpatients there is a need to develop accurate methods of diagnosing andtreatment of PAP, and the compositions and methods described herein canbe used in diagnosing and treating PAP.

Methods and Compositions

The methods described herein are effected by administering to a subjecta pharmaceutical compositions comprising a molecule capable of modifyinga tissue level and/or activity of at least one type of LOX to therebymodulate angiogenesis in the mammalian tissue. Administration may beinto a mammalian tissue. Modifying the tissue level and/or activity ofat least one type of LOX or LOXL can modulate angiogenesis, primarytumor development, tumor metastasis and/or PAP. Expression level oractivity of the LOX or LOXL can also be detected and used to diagnose acondition, such as PAP.

As used herein, the phrase “tissue level” refers to the level of LOX orLOXL protein present in the tissue at a given time point. At times, itmay be advantageous to measure tissue levels of the active forms of LOXor LOXL. Protein levels are determined by factors such as, transcriptionand/or translation rates, RNA or protein turnover and/or proteinlocalization within the cell. As such any molecule which effects any ofthese factors can modify the tissue level of LOX or LOXL.

As used herein the term “activity” refers to an enzymatic activity ofLOX or LOXL. A molecule which can modify the enzymatic activity maydirectly or indirectly alter substrate specificity of the enzyme oractivity of the catalytic site thereof.

There are numerous examples of compositions that can comprise moleculeswhich can specifically modify the tissue level and/or activity of alysyl oxidase. Such molecules can be categorized into lysyl oxidase“downregulators” or “upregulators.”

Downregulators

One example of an agent capable of downregulating a lysyl oxidaseprotein is an antibody or antibody fragment capable of specificallybinding lysyl oxidase or at least part of the lysyl oxidase protein(e.g., region spanning the catalytic site) and inhibiting its activitywhen introduced into the mammalian tissue. As such, an antibody or anantibody fragment directed at a lysyl oxidase can be used to suppress orarrest the formation of blood vessels, and to inhibit tumor fibrosis andmetastasis.

The antibody can specifically bind to at least one epitope of LOX orLOXL. As used herein, the term “epitope” refers to any antigenicdeterminant on an antigen to which the paratope of an antibody binds.

Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or carbohydrate side chainsand usually have specific three-dimensional structural characteristics,as well as specific charge characteristics.

As used herein, the term “antibody” includes intact molecules as well asfunctional fragments thereof, such as Fab, F(ab′)₂, and Fv that arecapable of binding to macrophages. These functional antibody fragmentsare defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule, can beproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule that can be obtained by treating wholeantibody with pepsin, followed by reduction, to yield an intact lightchain and a portion of the heavy chain; two Fab′ fragments are obtainedper antibody molecule; (3) (Fab′)₂, the fragment of the antibody thatcan be obtained by treating whole antibody with the enzyme pepsinwithout subsequent reduction; F(ab′)₂ is a dimer of two Fab′ fragmentsheld together by two disulfide bonds; (4) Fv, defined as a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains; and(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York 1988).

Antibody fragments according to the present disclosure can be preparedby proteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (such as in Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)₂. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, by Porter,Biochem. J. 73:119-126 (1959), and U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein. Other methods of cleavingantibodies, such as separation of heavy chains to form monovalentlight-heavy chain fragments, further cleavage of fragments, or otherenzymatic, chemical, or genetic techniques may also be used, so long asthe fragments bind to the antigen that is recognized by the intactantibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc. Nat.Acad. Sci. USA 69-2659-62 (1972). Alternatively, the variable chains canbe linked by an intermolecular disulfide bond or cross-linked bychemicals such as glutaraldehyde. The Fv fragments can comprise VH andVL chains connected by a peptide linker. These single-chain antigenbinding proteins (sFv) can be prepared by constructing a structural genecomprising DNA sequences encoding the VH and VL domains connected by anoligonucleotide. The structural gene is inserted into an expressionvector, which is subsequently introduced into a host cell such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFvs are described, for example, by Whitlow and Filpula, Methods2:97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al.,Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells, for example, as described inLarrick and Fry, Methods, 2:106-10 (1991).

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies), which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues form a CDR of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody comprises substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally can also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers(Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)),by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol., 227:381-388 (1991); Marks et al., J. Mol. Biol., 222:581-597(1991)). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al., J. Immunol., 147:86-95 (1991)). Similarly, humanantibodies can be made by introduction of human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and in the following scientific publications: Marks et al.,Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859(1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., NatureBiotechnology 14:845-851 (1996); Neuberger, Nature Biotechnology 14:826(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

As is described below, various approaches can be used to reduce orabolish transcription or translation of a lysyl oxidase.

Polynucleotides

One approach is the use of polynucleotides to downregulate theexpression or activity of LOX or LOXL. “Polynucleotide,” “nucleotide,”“nucleic acid,” and “oligonucleotide” are used interchangeably herein.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides maybe interrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. An oligonucleotide may be isolated, such that theoligonucleotide is separated from other constituents, cellular andotherwise, that in nature is normally associated with thepolynucleotide, peptide, polypeptide, protein, antibody, or fragmentsthereof.

Modified polynucleotides may be used in the present invention. Modifiedpolynucleotides can have improved half-life and/or membrane penetration.A large number of variations in polynucleotide backbones are known inthe arts. Oligonucleotides can be modified either in the base, the sugaror the phosphate moiety. These modifications include, for example, theuse of methylphosphonates, monothiophosphates, dithiophosphates,phosphoramidates, phosphate esters, bridged phosphorothioates, bridgedphosphoramidates, bridged methylenephosphonates, dephosphointernucleotide analogs with siloxane bridges, carbonate bridges,carboxymethyl ester bridges, carbonate bridges, carboxymethyl esterbridges, acetamide bridges, carbamate bridges, thioether bridges,sulfoxy bridges, sulfono bridges, various “plastic” DNAs, anomericbridges and borane derivatives, such as in Cook, Anti-Cancer Drug Design6: 585 (1991).

International patent application WO 89/12060 discloses various buildingblocks for synthesizing oligonucleotide analogs, as well asoligonucleotide analogs formed by joining such building blocks in adefined sequence. The building blocks may be either “rigid” (i.e.,containing a ring structure) or “flexible” (i.e., lacking a ringstructure). In both cases, the building blocks contain a hydroxy groupand a mercapto group, through which the building blocks are said to jointo form oligonucleotide analogs. The linking moiety in theoligonucleotide analogs is selected from the group consisting of sulfide(—S—), sulfoxide (—SO—), and sulfone (—SO2—).

International patent application WO 92/20702 describes an acyclicoligonucleotide which includes a peptide backbone on which any selectedchemical nucleobases or analogs are stringed and serve as codingcharacters as they do in natural DNA or RNA. These new compounds, knownas peptide nucleic acids (PNAs), are not only more stable in cells thantheir natural counterparts, but also bind natural DNA and RNA 50 to 100times more tightly than the natural nucleic acids cling to each other.PNA oligomers can be synthesized from the four protected monomerscontaining thymine, cytosine, adenine and guanine by Merrifieldsolid-phase peptide synthesis. In order to increase solubility in waterand to prevent aggregation, a lysine amide group can be placed at theC-terminal region.

A linear sequence or sequence is an order of nucleotides in apolynucleotide in a 5′ to 3′ direction in which residues that neighboreach other in the sequence are contiguous in the primary structure ofthe polynucleotide. A partial sequence is a linear sequence of part of apolynucleotide that is known to comprise additional residues in one orboth directions.

A linear sequence of nucleotides is identical to another linearsequence, if the order of nucleotides in each sequence is the same, andoccurs without substitution, deletion, or material substitution. It isunderstood that purine and pyrimidine nitrogenous bases with similarstructures can be functionally equivalent in terms of Watson-Crickbase-pairing; and the inter-substitution of like nitrogenous bases,particularly uracil and thymine, or the modification of nitrogenousbases, such as by methylation, does not constitute a materialsubstitution. An RNA and a DNA polynucleotide have identical sequenceswhen the sequence for the RNA reflects the order of nitrogenous bases inthe polyribonucleotides, the sequence for the DNA reflects the order ofnitrogenous bases in the polydeoxyribonucleotides, and the two sequencessatisfy the other requirements of this definition. Where one or both ofthe polynucleotides being compared is double-stranded, the sequences areidentical if one strand of the first polynucleotide is identical withone strand of the second polynucleotide.

A vector may comprise the polynucleotides of the present invention. Thevector, a nucleic acid molecule that is typically self-replicating,transfers an inserted nucleic acid molecule into and/or between hostcells. Vectors include those that function primarily for insertion ofDNA or RNA into a cell, replication of vectors that function primarilyfor the replication of DNA or RNA, and expression vectors that functionfor transcription and/or translation of the DNA or RNA. Also includedare vectors that provide more than one of the above functions. Anexpression vector is a polynucleotide which, when introduced into anappropriate host cell, can be transcribed and translated into apolypeptide(s). An expression system usually connotes a suitable hostcell comprised of an expression vector that can function to yield adesired expression product

Host cells into which a vector or polynucleotide of the invention, e.g.,an expression vector, or an isolated nucleic acid molecule of theinvention has been introduced, refers not only to the particular cellbut also to the progeny or potential progeny of such a cell. A host cellcan be any prokaryotic or eukaryotic cell. For example, host cells caninclude bacterial cells such as E. coli, insect cells, yeast cells, ormammalian cells (such as Chinese hamster ovary cells (CHO) or COScells). Other suitable host cells are known to those skilled in the art.Many methods for introducing nucleic acids into host cells, both in vivoand in vitro, are well known to those skilled in the art and include,without limitation, calcium phosphate precipitation, electroporation,heat shock, lipofection, microinjection, and viral-mediated nucleic acidtransfer. Delivery strategies are described in Luft, J. Mol. Med.76:75-76 (1998); Kronenwett et al., Blood 91:852-862 (1998); Rajur etal., Bioconjug. Chem. 8:935-940 (1997); Lavigne et al., Biochem.Biophys. Res. Commun. 237:566-571 (1997) and Aoki et al., Biochem.Biophys. Res. Commun. 231:540-545 (1997). Delivery of thepolynucleotides of the present invention can also be to subjects,including mammals. For example, polynucleotides of the present inventioncan be delivered or administered to mammalian tissues.

Polynucleotides of the present invention includes antisenseoligonucleotides, ribozymes, DNAzymes, siRNA molecules including shRNA,and triple helix forming oligonucleotides, to downregulate theexpression or activity of one or more lysyl oxidase.

Antisense Polynucleotides

According to one aspect, downregulation of LOX or LOXL levels oractivity can be effected using an antisense polynucleotide capable ofspecifically hybridizing with an mRNA transcript encoding LOX or LOXL,such as LOXL2.

Design of antisense molecules which can be used to efficientlydownregulate LOX or LOXL2 is typically effected while considering twoaspects factors used in the antisense approach. The first aspect isdelivery of the oligonucleotide into the cytoplasm of the appropriatecells, while the second aspect is design of an oligonucleotide whichspecifically binds the designated mRNA within cells in a way whichinhibits translation thereof.

Several considerations are typically taken into account when designingantisense oligonucleotides. For efficient in vivo inhibition of geneexpression using antisense oligonucleotides or analogs, theoligonucleotides or analogs typically fulfill the following requirements(i) sufficient specificity in binding to the target sequence; (ii)solubility in water; (iii) stability against intra- and extracellularnucleases; (iv) capability of penetration through the cell membrane; and(v) when used to treat an organism, low toxicity. Algorithms foridentifying those sequences with the highest predicted binding affinityfor their target mRNA based on a thermodynamic cycle that accounts forthe energy of structural alterations in both the target mRNA and theoligonucleotide are available, for example, as described in Walton etal. Biotechnol Bioeng 65:1-9 (1999).

Such algorithms have been successfully used to implement an antisenseapproach in cells. For example, the algorithm developed by Walton et al.enabled scientists to successfully design antisense oligonucleotides forrabbit β-globin (RBG) and mouse tumor necrosis factor-α (TNF α)transcripts. The same research group has also reported that theantisense activity of rationally selected oligonucleotides against threemodel target mRNAs (human lactate dehydrogenase A and B and rat gp130)in cell culture as evaluated by a kinetic PCR technique proved effectivein almost all cases, including tests against three different targets intwo cell types with phosphodiester and phosphorothioate oligonucleotidechemistries.

In addition, several approaches for designing and predicting efficiencyof specific oligonucleotides using an in vitro system are also published(Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)).

An antisense molecule which can be used with the present disclosureincludes a polynucleotide or a polynucleotide analog of at least 10bases, for example, between 10 and 15, between 15 and 20 bases, at least17, at least 18, at least 19, at least 20, at least 22, at least 25, atleast 30, or even at least 40 bases which is hybridizable in vivo, underphysiological conditions, with a portion of a polynucleotide strandencoding a polypeptide at least 50% homologous to SEQ ID NO:1, 4, 5 or 7or at least 75% homologous to an N-terminal portion thereof asdetermined using the BestFit software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap creationpenalty equals 8 and gap extension penalty equals 2.

The antisense oligonucleotides used by the present disclosure can beexpressed from a nucleic acid construct administered into the tissue, inwhich case inducible promoters can be used such that antisenseexpression can be switched on and off, or alternatively sucholigonucleotides can be chemically synthesized and administered directlyinto the tissue, as part of, for example, a pharmaceutical composition.

The ability of chemically synthesizing oligonucleotides and analogsthereof having a selected predetermined sequence offers means fordownmodulating gene expression. Four types of gene expression modulationstrategies may be considered.

At the transcription level, antisense or sense oligonucleotides oranalogs that bind to the genomic DNA by strand displacement or theformation of a triple helix, may prevent transcription. At thetranscript level, antisense oligonucleotides or analogs that bind targetmRNA molecules lead to the enzymatic cleavage of the hybrid byintracellular RNase H. In this case, by hybridizing to the targetedmRNA, the oligonucleotides or oligonucleotide analogs provide a duplexhybrid recognized and destroyed by the RNase H enzyme. Alternatively,such hybrid formation may lead to interference with correct splicing. Asa result, in both cases, the number of the target mRNA intacttranscripts ready for translation is reduced or eliminated.

At the translation level, antisense oligonucleotides or analogs thatbind target mRNA molecules prevent, by steric hindrance, binding ofessential translation factors (ribosomes), to the target mRNA, aphenomenon known in the art as hybridization arrest, disabling thetranslation of such mRNAs.

Unmodified oligonucleotides are typically impractical for use asantisense sequences since they have short in vivo half-lives, duringwhich they are degraded rapidly by nucleases. Furthermore, they areoften difficult to prepare in more than milligram quantities. Inaddition, such oligonucleotides are usually poor cell membranepenetrants. Thus, oligonucleotide analogs are usually devised in asuitable manner.

For example, problems arising in connection with double-stranded DNA(dsDNA) recognition through triple helix formation have been diminishedby a clever “switch back” chemical linking, whereby a sequence ofpolypurine on one strand is recognized, and by “switching back,” ahomopurine sequence on the other strand can be recognized. Also, goodhelix formation has been obtained by using artificial bases, therebyimproving binding conditions with regard to ionic strength and pH.

RNA oligonucleotides may also be used for antisense inhibition as theyform a stable RNA-RNA duplex with the target, suggesting efficientinhibition. However, due to their low stability, RNA oligonucleotidesare typically expressed inside the cells using vectors designed for thispurpose. This approach may be used when attempting to target an mRNAthat encodes an abundant and long-lived protein.

Antisense therapeutics can be used to treat many life-threateningdiseases with a number of advantages over traditional drugs. Traditionaldrugs typically intervene after a disease-causing protein is formed.Antisense therapeutics, however, can block mRNAtranscription/translation and intervene before a protein is formed, andsince antisense therapeutics target only one specific mRNA, they can bemore effective with fewer side effects than current protein-inhibitingtherapy.

Several clinical trials have demonstrated safety, feasibility andactivity of antisense oligonucleotides. For example, antisenseoligonucleotides suitable for the treatment of cancer have beensuccessfully used (Holmund et al., Curr. Opin. Mol. Ther. 1:372-385(1999)), while treatment of hematological malignancies via antisenseoligonucleotides targeting c-myb gene, p53 and Bcl-2 had enteredclinical trials and had been shown to be tolerated by patients (Gerwitz,Curr. Opin. Mol. Ther. 1: 297-306 (1999)).

More recently, antisense-mediated suppression of human heparanase geneexpression has been reported to inhibit pleural dissemination of humancancer cells in a mouse model (Uno et al., Cancer Res 61:7855-60(2001)).

The first antisense drug was recently approved by the FDA. The drug,Fomivirsen, was developed by Isis, and is indicated for local treatmentof cytomegalovirus in patients with AIDS who are intolerant of or have acontraindication to other treatments for CMV retinitis or who wereinsufficiently responsive to previous treatments for CMV retinitis(Pharmacotherapy News Network).

Thus, the current consensus is that recent developments in the field ofantisense technology which, as described above, have led to thegeneration of highly accurate antisense design algorithms and a widevariety of oligonucleotide delivery systems, enable an ordinarilyskilled artisan to design and implement antisense approaches suitablefor downregulating expression of known sequences without having toresort to undue trial and error experimentation.

Ribozyme

Another agent capable of downregulating a lysyl oxidase is a ribozymemolecule capable of specifically cleaving an mRNA transcript encoding aLOX or LOXL. Ribozymes are being increasingly used for thesequence-specific inhibition of gene expression by the cleavage of mRNAsencoding proteins of interest (Welch et al., Curr. Opin. Biotechnol.9:486-496 (1998)). The possibility of designing ribozymes to cleave anyspecific target RNA has rendered them valuable tools in both basicresearch and therapeutic applications. In the therapeutics area,ribozymes have been exploited to target viral RNAs in infectiousdiseases, dominant oncogenes in cancers and specific somatic mutationsin genetic disorders (Welch et al., Clin. Diagn. Virol. 10:163-171(1998)). Most notably, several ribozyme gene therapy protocols for HIVpatients are already in Phase 1 trials. More recently, ribozymes havebeen used for transgenic animal research, gene target validation andpathway elucidation. Several ribozymes are in various stages of clinicaltrials. ANGIOZYME was the first chemically synthesized ribozyme to bestudied in human clinical trials. ANGIOZYME specifically inhibitsformation of the VEGF-r (Vascular Endothelial Growth Factor receptor), akey component in the angiogenesis pathway. Ribozyme Pharmaceuticals,Inc., as well as other firms have demonstrated the importance ofanti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozymedesigned to selectively destroy Hepatitis C Virus (HCV) RNA, was foundeffective in decreasing Hepatitis C viral RNA in cell culture assays(Ribozyme Pharmaceuticals, Inc.).

DNAzynze

Another agent capable of downregulating a lysyl oxidase is a DNAzymemolecule capable of specifically cleaving an mRNA transcript or DNAsequence of LOX or LOXL. DNAzymes are single-stranded polynucleotideswhich are capable of cleaving both single and double stranded targetsequences (Breaker and Joyce, Chemistry and Biology, 2:655-660 (1995);Santoro and Joyce, Proc. Natl. Acad. Sci. USA, 943:4262-4266 (1997)). Ageneral model (the “10-23” model) for the DNAzyme has been proposed.“10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides,flanked by two substrate-recognition domains of seven to ninedeoxyribonucleotides each. This type of DNAzyme can effectively cleaveits substrate RNA at purine:pyrimidine junctions (Santoro and Joyce,Proc. Natl. Acad. Sci. USA, 943:4262-4266 (1997); Khachigian, Curr.Opin. Mol. Ther. 4:119-121 (2002)).

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174. DNAzymes of similardesign directed against the human Urokinase receptor were recentlyobserved to inhibit Urokinase receptor expression, and successfullyinhibit colon cancer cell metastasis in vivo (Itoh et al., 2002,Abstract 409, Ann Meeting Am. Soc. Gen. Ther. www.asgt.org). In anotherapplication, DNAzymes complementary to bcr-abl oncogenes were successfulin inhibiting the oncogenes expression in leukemia cells, and lesseningrelapse rates in autologous bone marrow transplant in cases of CML andALL.

siRNA

Another mechanism of down regulating a lysyl oxidase at the transcriptlevel is RNA interference (RNAi), an approach which utilizes smallinterfering dsRNA (siRNA or small hairpin RNA, shRNA) molecules that arehomologous to the target mRNA and lead to its degradation (Carthew,Curr. Opin. Cell. Biol. 13: 244-248 (2001)). For example, infection ofdiverse types of cancer cells with expression of a LOXL2 specific shRNAis effective in altering both their morphology and invasiveness (Example1).

RNA interference is typically a two-step process. In the first step,which is termed as the initiation step, input dsRNA is digested into21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by theaction of Dicer, a member of the RNase III family of dsRNA-specificribonucleases, which processes (cleaves) dsRNA (introduced directly orvia a transgene or a virus) in an ATP-dependent manner. Successivecleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each with2-nucleotide 3′ overhangs (Hutvagner and Zamore, Curr. Opin. Genet. Dev.12: 225-232 (2002); Bernstein, Nature 409:363-366 (2001)).

In the effector step, the siRNA duplexes bind to a nuclease complex toform the RNA-induced silencing complex (RISC). An ATP-dependentunwinding of the siRNA duplex is required for activation of the RISC.The active RISC then targets the homologous transcript by base pairinginteractions and typically cleaves the mRNA into approximately 12nucleotide fragments from the 3′ terminus of the siRNA (Hutvagner andZamore, Curr. Opin. Genet. Dev. 12: 225-232 (2002); Hammond et al., Nat.Rev. Gen. 2:110-119 (2001); Sharp, Genes. Dev. 15:485-490 (2001)).Although the mechanism of cleavage is still to be elucidated, researchindicates that each RISC contains a single siRNA and an RNase (Hutvagnerand Zamore, Curr. Opin. Genet. Dev. 12: 225-232 (2002)).

Because of the remarkable potency of RNAi, an amplification step withinthe RNAi pathway has been suggested. Amplification could occur bycopying of the input dsRNAs which would generate more siRNAs, or byreplication of the siRNAs formed. Alternatively or additionally,amplification could be effected by multiple turnover events of the RISC(Hutvagner and Zamore, Curr. Opin. Genet. Dev. 12: 225-232 (2002);Hammond et al., Nat. Rev. Gen. 2:110-119 (2001); Sharp, Genes. Dev.15:485-490 (2001)). RNAi is also described in Tuschl, Chem. Biochem. 2:239-245 (2001); Cullen, Nat. Immunol. 3:597-599 (2002); and Brand,Biochem. Biophys. Act. 1575:15-25 (2002).

Synthesis of RNAi molecules suitable for use with the present disclosurecan be effected as follows. First, the LOX or LOXL mRNA sequence isscanned downstream of the AUG start codon for AA dinucleotide sequences.Occurrence of each AA and the 3′ adjacent 19 nucleotides is recorded aspotential siRNA target sites. The siRNA target sites are selected fromthe open reading frame, as untranslated regions (UTRs) are richer inregulatory protein binding sites. UTR-binding proteins and/ortranslation initiation complexes may interfere with binding of the siRNAendonuclease complex (Tuschl, Chem. Biochem. 2: 239-245 (2001)). It willbe appreciated though, that siRNAs directed at untranslated regions mayalso be effective, as demonstrated for GAPDH wherein siRNA directed atthe 5′ UTR mediated about 90% decrease in cellular GAPDH mRNA andcompletely abolished protein level(www.ambion.com/techlib/tn/91/912.html). Second, potential target sitesare compared to an appropriate genomic database (e.g., human, mouse, ratetc.) using any sequence alignment software, such as the BLAST softwareavailable from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/). Putativetarget sites which exhibit significant homology to other codingsequences are filtered out.

Qualifying target sequences are selected as template for to siRNAsynthesis. Selected sequences can include those with low G/C content asthese have been shown to be more effective in mediating gene silencingas compared to those with G/C content higher than 55%. Several targetsites can be selected along the length of the target gene forevaluation. For better evaluation of the selected siRNAs, a negativecontrol is used in conjunction. Negative control siRNA can include thesame nucleotide composition as the siRNAs but lack significant homologyto the genome. Thus, a scrambled nucleotide sequence of the siRNA may beused, provided it does not display any significant homology to any othergene.

The siRNA molecules of the present disclosure can be transcribed fromexpression vectors which can facilitate stable expression of the siRNAtranscripts once introduced into a host cell. These vectors areengineered to express shRNAs, which are processed in vivo into siRNAmolecules capable of carrying out gene-specific silencing (Brummelkampet al., Science 296:550-553 (2002); Paddison et al., Genes Dev.16:948-958 (2002); Paul et al., Nature Biotech. 20: 505-508 (2002); Yuet al., Proc. Natl. Acad. Sci. USA 99:6047-6052(2002)).

ShRNAs are single-stranded polynucleotides with a hairpin loopstructure. The single-stranded polynucleotide has a loop segment linkingthe 3′ end of one strand in the double-stranded region and the 5′ end ofthe other strand in the double-stranded region. The double-strandedregion is formed from a first sequence that is hybridizable to a targetsequence, such as a polynucleotide encoding LOXL2, or a LOXL2 mRNA, anda second sequence that is complementary to the first sequence, thus thefirst and second sequence form a double stranded region to which thelinking sequence connects the ends of to form the hairpin loopstructure. The first sequence can be hybridizable to any portion of apolynucleotide encoding LOXL2. The double-stranded stem domain of theshRNA comprises a restriction endonuclease site.

The stem-loop structure of shRNAs can have optional nucleotideoverhands, such as 2-bp overhands, for example, 3′ UU-overhangs. Whilethere may be variation, stems typically range from approximately 15 to49, approximately 15 to 35, approximately 19 to 35, approximately 21 to31 bp, or approximately 21 to 29 bp, and the loops can range fromapproximately 4 to 30 bp, for example, about 4 to 23 bp.

For expression of shRNAs within cells, plasmid vectors containing eitherthe polymerase III H1-RNA or U6 promoter, a cloning site for thestem-looped RNA insert, and a 4 5-thymidine transcription terminationsignal can be employed. The Polymerase III promoters generally havewell-defined initiation and stop sites and their transcripts lackpoly(A) tails. The termination signal for these promoters is defined bythe polythymidine tract, and the transcript is typically cleaved afterthe second uridine. Cleavage at this position generates a 3′ UU overhangin the expressed shRNA, which is similar to the 3′ overhangs ofsynthetic siRNAs. Additional methods for expressing the shRNA inmammalian cells are described in the references cited above.

An example of a suitable expression vector is the pSUPER™, whichincludes the polymerase-III H1-RNA gene promoter with a well definedstart of transcription and a termination signal consisting of fivethymidines in a row (T5) (Brummelkamp et al., Science 296:550-553(2002)). The cleavage of the transcript at the termination site is at asite following the second uridine, thus yielding a transcript whichresembles the ends of synthetic siRNAs, which also contain nucleotideoverhangs. siRNA is cloned such that it includes the sequence ofinterest, i.e., LOX or LOXL separated by a short spacer from the reversecomplement of the same sequence. The resulting transcript folds back onitself to form a stem-loop structure, which mediates LOX or LOXL RNAi.For example, sequences that comprise a DNA sequence encoding the shRNAfor LOXL2, such as SEQ ID NO: 20 (sh.LOXL2.197 or si-197), or SEQ ID NO:21(sh.LOXL2.195, or si-195) may mediate LOXL2 RNAi. The sequences thatmediate LOXL2 RNAi may also comprise SEQ ID NO: 22, 23, 24, 25, 26, orportions thereof.

Another suitable siRNA expression vector encodes the sense and antisensesiRNA under the regulation of separate polIII promoters (Miyagishi andTaira, Nature Biotech. 20:497-500 (2002)). The siRNA, generated by thisvector also includes a five thymidine (T5) termination signal.

Since approaches for introducing synthetic siRNA into cells bylipofection can result in low transfection efficiencies in some celltypes and/or short-term persistence of silencing effects, vectormediated methods have been developed.

Thus, siRNA molecules utilized by the present disclosure can bedelivered into cell using retroviruses. Delivery of siRNA usingretroviruses provides several advantages over methods, such aslipofection, since retroviral delivery typically is more efficient,uniform and immediately selects for stable “knock-down” cells (Devroeand Silver, BMC Biotechnol. 2:15 (2002)).

Recent scientific publications have validated the efficacy of such shortdouble stranded RNA molecules in inhibiting target mRNA expression andthus have clearly demonstrated the therapeutic potential of suchmolecules. For example, RNAi has been utilized to inhibit expression ofhepatitis C (McCaffrey et al., Nature 418:38-39 (2002)), HIV-1 (Jacqueet al., Nature 418:435-438 (2002)), cervical cancer cells (Jiang andMilner, Oncogene 21:6041-6048 (2002)) and leukemic cells (Wilda et al.,Oncogene 21, 5716-5724 (2002)).

Triple Helix Forming Oligonucleotides (TFO)

An additional method of regulating the expression of LOX or LOXL incells is via triplex forming oligonucleotides (TFOs). Recent studieshave shown that TFOs can be designed which can recognize and bind topolypurine/polypyrimidine regions in double-stranded helical DNA in asequence-specific manner. These recognition rules are outlined by MaherIII et al., Science 245:725-730 (1989); Moser et al., Science238:645-630 (1987); Beal et al., Science 251:1360-1363 (1992); Cooney etal., Science 241:456-459 (1988); and Hogan et al., EP Publication375408. Modification of the oligonucleotides, such as the introductionof intercalators and backbone substitutions, and optimization of bindingconditions (pH and cation concentration) have aided in overcominginherent obstacles to TFO activity such as charge repulsion andinstability, and it was recently shown that synthetic oligonucleotidescan be targeted to specific sequences (see Seidman and Glazer, J. Clin.Invest. 112:487-494 (2003)).

In general, the triplex-forming oligonucleotide has the sequencecorrespondence:

oligo 3′--A G G T duplex 5′--A G C T duplex 3′--T C G A

However, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch, BMC Biochem,2002, Sept12, Epub). The same authors demonstrated that TFOs designedaccording to the A-AT and G-GC rule do not form non-specific triplexes,indicating that the triplex formation is sequence specific.

Thus for any given sequence in the LOX or LOXL regulatory region, atriplex forming sequence may be devised. Triplex-formingoligonucleotides can be at least 15, 25, 30 or more nucleotides inlength. They can also be up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs,and formation of the triple helical structure with the target DNAinduces steric and functional changes, blocking transcription initiationand elongation, allowing the introduction of desired sequence changes inthe endogenous DNA and resulting in the specific downregulation of geneexpression. Examples of such suppression of gene expression in cellstreated with TFOs include knockout of episomal supFG1 and endogenousHPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res.27:1176-1181 (1999); Puri et al., J. Biol. Chem. 276: 28991-28998(2001)), and the sequence and target specific downregulation ofexpression of the Ets2 transcription factor, important in prostatecancer etiology (Carbone et al., Nucl. Acid Res. 31:833-843 (2003)), andthe pro-inflammatory ICAM-1 gene (Besch et al., J. Biol. Chem.277:32473-32479 (2002)). In addition, Vuyisich and Beal have shown thatsequence specific TFOs can bind to dsRNA, inhibiting activity ofdsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich andBeal, Nuc. Acids Res. 28: 2369-2374(2000)).

Additionally, TFOs designed according to the abovementioned principlescan induce directed mutagenesis capable of effecting DNA repair, thusproviding both downregulation and upregulation of expression ofendogenous genes (Seidman and Glazer, J. Clin. Invest. 112:487-494(2003)). Detailed description of the design, synthesis andadministration of effective TFOs can be found in U.S. Patent ApplicationNos. 2003/017068 and 2003/0096980 to Froehler et al, and 2002 0128218and 2002 0123476 to Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.

The downregulators described hereinabove are useful for inhibitingangiogenesis in tumor tissue. It has been shown that PF4, a lysyloxidase binding protein which inhibits angiogenesis in tumor tissuespecifically accumulates in newly formed blood vessels of tumors(angiogenic vessels) but not in established blood vessels (Hansell etal., Amer. J. Physiol-Heart. Circ. Phy. 38:H829-H836 (1995); Reiser etal., FASEB J. 6: 2439-2449 (1992)).

Newly formed angiogenic blood vessels are typically more permeable toproteins than established blood vessels because the major inducer ofangiogenesis in many angiogenic diseases is VEGF, a growth factor whichalso functions as a potent blood vessel permeabilizing factor (VPF)(Neufeld et al., FASEB J. 13:9-22 (1999)). Tumor associated bloodvessels are therefore typically in a permanent state ofhyperpermeability due to deregulated over-expression of VEGF and assuch, a downregulator molecule used by the method of the presentdisclosure could be able to extravasate efficiently from tumor bloodvessels but much less efficiently from normal stabilized blood vessels.

Upregulators

Several approaches can be utilized to increase the levels of LOX or LOXLand as such to enhance the formation of blood vessels.

For example, a nucleic acid construct including a constitutive,inducible or tissue specific promoter positioned upstream of apolynucleotide encoding a polypeptide having LOX or LOXL activity, suchas the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9 can beadministered into a mammalian tissue. The LOX or LOXL expressed fromthis construct could substantially increase the levels of LOX or LOXLwithin the cells of the tissue and as such enhance angiogenesis.

The polynucleotide segments encoding the LOX or LOXL can be ligated intoa commercially available expression vector. Such an expression vectorincludes a promoter sequence for directing transcription of thepolynucleotide sequence in the cell in a constitutive or induciblemanner. A suitable promoter can be, for example, a Tie-2 promoter whichis capable of directing lysyl oxidase specific gene expression inendothelial cells (see Schlaeger et al., Proc. Natl. Acad. Sci. U.S.A.94, 3058-3063 (1997)). The expression vector of the present disclosurecan further include additional polynucleotide sequences such as forexample, sequences encoding selection markers or reporter polypeptides,sequences encoding origin of replication in bacteria, sequences thatallow for translation of several proteins from a single mRNA such as aninternal ribosome entry site (IRES), sequences for genomic integrationof the promoter-chimeric polypeptide encoding region and/or sequencesgenerally included in mammalian expression vector such as pcDNA3,pcDNA3.1(+/−), pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto,pCMV/myc/cyto, pCR3.1, which are available from Invitrogen, pCI which isavailable from Promega, pBK-RSV and pBK-CMV which are available fromStrategene, pTRES which is available from Clontech, and theirderivatives.

It will be appreciated that such commercially available vector systemscan easily be modified via commonly used recombinant techniques in orderto replace, duplicate or mutate existing promoter or enhancer sequencesand/or introduce any additional polynucleotide sequences.

An agent capable of upregulating a LOX or LOXL may also be any compoundwhich is capable of increasing the transcription and/or translation ofan endogenous DNA or mRNA encoding a LOX or LOXL using for example gene“knock in” techniques.

Enhancer elements can be “knocked in” adjacent to endogenous lysyloxidase coding sequences to thereby increase transcription therefrom.

Further details relating to the construction and use of knock-out andknock-in constructs is provided elsewhere (Fukushige and Ikeda, DNA Res.3:73-80 (1996); Bedell et al., Genes Dev. 11:1-11 (1997); Bermingham etal., Genes Dev. 10:1751-1762 (1996)).

It will be appreciated that direct administration of a polypeptideexhibiting a LOX or LOXL activity can also be utilized for enhancingangiogenesis.

Thus, affinity binding assays and/or activity assays, the principles ofwhich are well known in the art, can be used to screen for novelcompounds (e.g., substrate analogs) which can specifically regulate theactivity of a lysyl oxidase and as such can be used with the presentinvention.

An assay suitable for use with this aspect of the present disclosure hasbeen previously described in a study conducted by Bedell-Hogan et al., JBiol Chem. 268:10345-10350 (1993).

Administration

Previous studies correlated expression levels of LOXL2 to the metastaticproperties of breast cancer derived cell lines, indicating that LOXL2may play additional roles in tumor invasiveness in addition to its rolein angiogenesis.

Thus, the present disclosure provides a method of inhibiting metastasisand/or fibrosis in a mammalian tissue using compositions describedherein. The method is effected by administering to the mammalian tissuea molecule capable of downregulating a tissue level and/or an activityof at least one type of a lysyl oxidase, such as shRNA disclosed herein.

The method of the present disclosure can be used to treat human patientsthat have been diagnosed with cancerous tumors, by administering any ofthe downregulating molecules described herein above, in order to reducethe tissue level and/or activity of at least one type of a lysyloxidase.

As used herein, the phrase “cancerous tumor” refers to any malignanttumor within a human body including, but not limiting to, tumors withmetastases. In addition, and without being bound to any particular typeof cancerous tumor, the present disclosure is useful to treat breastcancer tumors, with or without metastases.

As used herein, the phrase “administering” refers to all modes ofadministration described herein below with respect to the pharmaceuticalcompositions of the present invention. Administration also refers to allmodes of administration described herein below with respect to anyagent, including polynucleotides of the present invention, for atherapeutic effect. Administration may be of an amount effective to havea therapeutic effect. The therapeutic effect may be for treating orinhibiting a condition or disorder, such as cancerous tumors, primary ormetatstatic, PAP, as well as disorders or conditions associated withfibrosis, and/or angiogenesis. An effective amount as used herein refersthe amount or dosage of that composition, such as an agent, includingpolynucleotides of the present invention that is required to induce adesired effect. An effective amount amount of a pharmaceuticalcomposition, or of an agent, such as a polynucleotide is meant to be anontoxic but sufficient amount of the agent or composition, to providethe desired effect, i.e., inhibiting, preventing, or reversing, theonset or progressive course of a cancer, primary or metatstatic, PAP,inflammation, and/or conditions or disorders related to fibrosis orangiogenesis.

Administration includes, but is not limited to, local administration atthe tumor tissue, an organ where the cancerous tumor was diagnosedand/or related tissues that typically form metastases (Hortobagyi,Semin. Oncol. 29: 134-144 (2002); Morrow and Gradishar, BMJ 324:410-414(2002)). Examples of related tissue include lymph nodes adjacent to, forexample, breast tissue and bones.

Administration can also be effected in a systemic manner in order totreat the affected tissue, i.e., the tissue where the cancerous tumorwas formed and where metastases are present or likely to be formed withtumor progression. A therapeutically effective amount of compositionsdescribed herein, may be administered, in which the amount is nontoxicbut sufficient to provide the desired effect, i.e., inhibiting,preventing, or reversing the onset or progressive course of a conditiondescribed here, including primary tumor formation or growth, metastasis,fibrosis, angiogenesis, and PAP.

Since any molecule capable of downregulating a lysyl oxidase activitycan be utilized by the methods described hereinabove, the presentdisclosure also provides a method of identifying molecules capable ofinhibiting metastasis and/or fibrosis.

This method is effected by screening and identifying molecules whichexhibit specific reactivity with at least one type of lysyl oxidase andtesting a metastasis and/or fibrosis inhibitory potential of thesemolecules.

Numerous types of molecules can be screened for reactivity with at leastone type of lysyl oxidase, examples include, but are not limited to,molecules such as antisense oligonucleotides, siRNA, DNAzymes, ribozymesand triple helix forming oligonucleotides (TFOs) that interact with apolynucleotide expressing a lysyl oxidase activity or molecules such asantibodies that interact with polypeptides having a lysyl oxidaseactivity. In addition, short peptides and other small molecules can alsobe screened by this method and used in the compositions and methods oftreatments disclosed herein.

Screening for cross reactivity can be effected by lysyl oxidaseenzymatic activity assays, by binding assays and the like. Examples ofsuitable assays are provided in Rodriguez et al., Arterioscler. Thromb.Vasc. Biol. 22:1409-1414 (2002); Wilson and Nock, Curr. Opin. Chem.Biol. 6:81-85 (2002); Uetz, Curr. Opin. Chem. Biol. 6: 57-62 (2002);Stoll et al., Front Biosci. 7:c13-32(2002)).

Testing a metastatic phenotype of transformed tumor cells can beperformed in vitro since nearly all steps of the metastatic process,including attachment, matrix degradation and migration, can be modeledexperimentally in vitro by measuring invasion of a reconstitutedbasement membrane (RBM). Metastatic invasiveness of tumor cell can bemodeled by migration of tumor cells into reconstituted basement membrane(RBM) in the presence and absence of a chemoattractant, such asfibroblast conditioned medium (FCM). The assay determines cells thathave attached to the RBM, degraded the RBM enzymatically and, finally,cells that have penetrated the FCM side of the membrane.

Since in vitro metastasis events correspond to steps observed in themetastatic spread of tumor cells through the basement membrane in vivo,in vitro invasiveness of cells can be assayed by the methods describedin Albini et al., Cancer Res. 47:3239-3245 (1987). Invasiveness assaysand other methods for assessing metastatic affects, are described inLeyton et al., Cancer Res. 54:3696-3699 (1994). Reconstituted basementmembrane preparations for use in accordance with the hereinabovedescribed assays are readily available from numerous commercialsuppliers. One such example membrane in this regard is “MATRIGEL”available from Collaborative Biomedical Products of Bedford, Mass.

In vitro evaluation of tumor cell metastatic phenotype can also beeffected by determining level and pattern of expression of one or moremetastasis associated markers such protease markers, which areconsidered to be an integral part of tumor metastasis (see U.S. Pat. No.6,303,318). One example is the arachidonic acid, the release of which incells can serve to indicate metastatic potential of a tumor (U.S. Pat.No. 6,316,416). In this regard, determining phospholipase A-2 (PLA2)activity, and the activity or abundance of factors that affect theactivity of PLA2, such as uteroglobin protein (U.S. Pat. No. 6,316,416)can serve as an indication of metastatic potential.

Determining pattern and level of expression of metastasis-associatedmarkers can be effected by one of several methods known in the art.

The presence or level of proteins indicative of metastatic potential oftumors can be determined in cells by conventional methods well known tothose of skill in the art. For instance, the techniques for making andusing antibody and other immunological reagents and for detectingparticular proteins in samples using such reagents are described inColigan et al. (Eds.), Current Protocols in Immunology, John Wiley &Sons, New York (1995), which is incorporated by reference herein inparts pertinent to making and using reagents useful for determiningspecific proteins in samples. As another example, immunohistochemicalmethods for determining proteins in cells in tissues are described inAusubel et al., (Eds.), Current Protocols in Molecular Biology, Volume2, Chapter 14, John Wiley & Sons, Inc. (1994), which is incorporated byreference herein in part pertinent to carrying out such determinations.Finally, Linnoila et al., A.J.C.P. 97: 235-243 (1992) and Peri et al.,J. Clin. Invest. 92: 2099-2109 (1992), incorporated herein as referredto above, describe techniques that may be used.

Metastatic potential can also be determined in vivo at the mRNA level.The presence and/or level of mRNA transcripts can be determined by avariety of methods known to those of skill in the art. A given mRNA maybe detected in cells by hybridization to a specific probe. Such probesmay be cloned DNAs or fragments thereof, RNA, typically made by in vitrotranscription, or oligonucleotide probes, usually generated by solidphase synthesis. Methods for generating and using probes suitable forspecific hybridization are well known and used in the art.

A variety of controls may be usefully employed to improve accuracy inmRNA detection assays. For instance, samples may be hybridized to anirrelevant probe and treated with RNAse A prior to hybridization, toassess false hybridization.

In order to modulate angiogenesis or inhibit metastasis or tumorfibrosis, the molecules used by the present disclosure can beadministered to the individual per se, or in a pharmaceuticalcomposition where it is mixed with suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration/targeting of a compound to a mammal.

As used herein the term “active ingredients” refers to the preparationaccountable for the biological effect, i.e. theupregulator/downregulator molecules used by the present disclosure tomodulate angiogenesis and the downregulators molecules used by thepresent disclosure to inhibit metastasis and tumor fibrosis.

The phrases “physiologically acceptable carrier” and “pharmaceuticallyacceptable carrier” are interchangeably used to refer to a carrier, suchas, for example, a liposome, a virus, a micelle, or a protein, or adiluent which do not cause significant irritation to the mammal and donot abrogate the biological activity and properties of the activeingredient. An adjuvant is included under these phrases.

The term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients, includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of compositions may befound in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa., latest edition.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, transnasal, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active ingredient with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the activeingredient of the invention to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for oral ingestion by a patient. Pharmacological preparations for oraluse can be made using a solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The preparation of the present disclosure may also be formulated inrectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

Pharmaceutical compositions suitable for use in context of the presentdisclosure include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose.

The pharmaceutical composition may form a part of an article ofmanufacturing which also includes a packaging material for containingthe pharmaceutical composition and a leaflet which provides indicationsof use for the pharmaceutical composition.

Thus, the present disclosure provides methods and pharmaceuticalcompositions useful modulating angiogenesis.

Such modulation activity can be used to treat arthritis (Koch, ArthritisRheum. 41:951-962 (1998); Paleolog and Fava, Springer Semin.Immunopathol. 20: 73-94 (1998)), diabetic retinopathy (Miller et al.,Diabetes Metab. Rev. 13:37-50 (1997)), psoriasis, (Detmar et al., J.Exp. Med. 180:1141-1146 (1994); Creamer et al., Br. J. Dermatol. 136,859-865(1997)) or vasculitis (Lie, Curr. Opin. Rheumatol. 4:47-55(1992); Klipple and Riordan, Rheum. Dis. Clin. North Am. 15:383-398(1989)).

In addition, the present disclosure also provides methods to treatdisease characterized by fragile blood vessels, including Marfanssyndrome, Kawasaki, Ehlers-Danlos, cutis-laxa, and takysu (Lie, Curr.Opin. Rheumatol. 4:47-55 (1992); Klipple and Riordan, Rheum. Dis. Clin.North Am. 15:383-398 (1989); Brahn et al., Clin. Immunol. Immunopathol.90:147-151 (1999); Cid et al., J. Clin. Invest. 91:977-985 (1993);Hoffman et al., Arthritis Rheum. 34:1466-1475 (1991)). It is possiblethat some of these diseases result from reduced or abolished lysyloxidase activity which leads to the synthesis of a fragile extracellularmatrix, and consequently, fragile blood vessels. As such, administrationof LOX or LOXL encoding sequences or polypeptides can be used to correctsome of the manifestations of these diseases.

The present disclosure also provides methods to treat diseases which arecharacterized by changes in the wall of blood vessels. For example,restenosis which is a common complication following balloon therapy,fibromuscular dysplasia (Begelman and Olin, Curr. Opin. Rheumatol.12:41-47 (2000)) and aortic stenosis (Palta et al., Circulation 101:2497-2502 (2000)) are all potentially treatable by the compositions andmethods described herein.

Diagnostics

In addition, LOXL2 is more highly expressed in metastatic tumors andcell lines than in non-metastatic tumors and cell lines. This suggeststhat levels of LOXL2 expression can be used as a diagnostic tool todetermine the malignancy of cancer cells, as well as, to determine andimplement suitable treatment regimens. LOXL2 and LOXL3 are also morehighly expressed in PAP, and thus levels of LOXL2, LOXL3, or both, canbe used as a diagnostic tool to determine PAP and implement suitabletreatment regimens. Detection agents, such as an antibody, a smallmolecule, antisense molecule, ribozyme, DNAzyme, triple helix formingoligonucleotides, siRNA, or shRNA can be used to assess the level oractivity of LOXL2, LOXL3, or both, in subjects.

Colon cancer is a highly treatable and often a curable disease whenlocalized to the bowel. However, in many cases, due to mis-diagnosis, apre-malignant colon hyperplasia progress into colon adenoma whichfurther develop into more malignant forms of low-grade and high-gradecolon adenocarcinoma. Once an individual is diagnosed with colon cancerthe malignancy of the tumor needs to be assessed in order to select forsuitable treatment regimens. The current practice for assessing themalignancy of a colon tumor is based on the tumor-node-metastases (TNM)staging system developed by the American Joint Committee on Cancer(AJCC). According to this method staging is based on scoring for thepresence or absence of cancerous cells in the tumor itself, in thesubmucosa of the bowel wall, in the muscular layer of the bowel wall(muscularis propria), and/or in the subserosa, pericolic or perirectaltissues, as well as in regional lymph nodes and distance metastases.Thus, staging of colon tumors involves multiple tissue biopsies andcomplex pathological evaluations which are time consuming and can resultin misdiagnosis.

LOXL2 expression in epithelial and/or connective tissue cells in a colontissue is indicative of a malignant colon cancer and thus provides a newmethod of assessing a malignancy of colon cancer tumors devoid of theabove limitations.

The expression of LOXL2 is correlated with the formation of benign colontumors and is increased in more malignant forms of colon cancer tumorsthus suggesting the use of LOXL2 in determining the stage of coloncancer tumors.

Thus according to another aspect of the present disclosure there isprovided a method of assessing a malignancy of a colon tumor. The methodis effected by determining a tissue level and/or an activity level of apolypeptide at least 75% homologous to the polypeptide set forth in SEQID NO: 2 or 9 in the colon tumor tissue, thereby assessing themalignancy of the colon tumor.

As is used herein, the phrase “assessing a malignancy of a to colontumor” refers to determining the stage of the colon tumor, i.e., theprogress of the colon tumor from a benign colon tumor to a highlymalignant colon cancer which invades the surrounding tissue.

The polypeptide detected by the present disclosure can be at least 75%,at least 80%, at least 85%, at least 90%, or at least 95% homologous toSEQ ID NO: 2 or 9, as determined using the BestFit software of theWisconsin sequence analysis package, utilizing the Smith and Watermanalgorithm, where gap creation penalty equals 8 and gap extension penaltyequals 2.

In some embodiments, the polypeptide is LOXL2 (SEQ ID NO: 2), a memberof the lysyl oxidase family which are fully described herein.

According to the methods described herein, a colon tumor tissue isobtained using a colon biopsy and/or a colon surgery using methods knowin the art. Once obtained, the tissue level and/or activity level of thepolypeptide of the present disclosure is determined in the colon tumortissue.

Similarly, for PAP diagnosis, a lung tissue sample can be obtained bylung biopsy and other methods known in the art. The polypeptide detectedby the present disclosure can be at least 75%, at least 80%, at least85%, at least 90%, or at least 95% homologous to SEQ ID NO: 2 or 9, asdetermined using the BestFit software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap creationpenalty equals 8 and gap extension penalty equals 2. The polypeptide ofthe present disclosure can be LOXL2 (SEQ ID NO: 2) or LOXL3 (SEQ ID NO.9), members of the lysyl oxidase family which are fully describedherein. The mRNA expression can also be detected and used for diagnosis.Furthermore, both LOXL2 and LOXL3 can be detected, either using the samedetection agent (for example an antibody that detects both proteins), ordifferent detection agents (for example an antibody that is specific forLOXL2 and another antibody specific for LOXL3; or different Northernprobes).

Determination of the tissue level of the polypeptides described hereinmay be accomplished directly using immunological methods.

The immunological detection methods used in context of the presentdisclosure are fully explained in, for example, Lane (Ed.), UsingAntibodies: A Laboratory Manual, Ed Harlow, Cold Spring HarborLaboratory Press (1999) and those familiar with the art will be capableof implementing the various techniques summarized hereinbelow as part ofthe present invention. All of the immunological techniques requireantibodies specific to at least one epitope of the polypeptide of thepresent invention. Immunological detection methods suited for use aspart of the present disclosure include, but are not limited to,radio-immunoassay (RIA), enzyme linked immunosorbent assay (ELISA),western blot, immunohistochemical analysis.

Radio-immunoassay (RIA): In one version, this method involvesprecipitation of the desired substrate, e.g., LOXL2, with a specificantibody and radiolabelled antibody binding protein (e.g., protein Alabeled with I¹²⁵) immobilized on a precipitable carrier such as agarosebeads. The number of counts in the precipitated pellet is proportionalto the amount of substrate.

In an alternate version of the RIA, a labeled substrate and an unlabeledantibody binding protein are employed. A sample containing an unknownamount of substrate is added in varying amounts. The decrease inprecipitated counts from the labeled substrate is proportional to theamount of substrate in the added sample.

Enzyme linked immunosorbent assay (ELISA): This method involves fixationof a sample (e.g., fixed cells or a proteinaceous solution) containing aprotein substrate (e.g., LOXL2) to a surface such as a well of amicroliter plate. A substrate specific antibody coupled to an enzyme isapplied and allowed to bind to the substrate. Presence of the antibodyis then detected and quantitated by a colorimetric reaction employingthe enzyme coupled to the antibody. Enzymes commonly employed in thismethod include horseradish peroxidase and alkaline phosphatase. If wellcalibrated and within the linear range of response, the amount ofsubstrate present in the sample is proportional to the amount of colorproduced. A substrate standard is generally employed to improvequantitative accuracy.

Western blot analysis: This method involves separation of a substrate(e.g., LOXL2) from other proteins by means of an acrylamide gel followedby transfer of the substrate to a membrane (e.g., nylon or PVDF).Presence of the substrate is then detected by antibodies specific to thesubstrate, which are in turn detected by antibody binding reagents.Antibody binding reagents may be, for example, protein A, or otherantibodies. Antibody binding reagents may be radiolabelled or enzymelinked as described hereinabove. Detection may be by autoradiography,colorimetric reaction or chemiluminescence. This method allows bothquantitation of an amount of substrate and determination of its identityby a relative position on the membrane which is indicative of amigration distance in the acrylamide gel during electrophoresis.

Immunohistochemical analysis: This method involves detection of asubstrate in situ in fixed tissue by substrate specific antibodies. Thesubstrate specific antibodies may be enzyme linked or linked tofluorophores and detected by microscopy and subjective evaluation. Ifenzyme linked antibodies are employed, a colorimetric reaction may beemployed.

Since tissue levels of a polypeptide can be inferred from the levels ofmRNA encoding such a polypeptide, the method according to this aspect ofthe present disclosure can also employ various polynucleotide detectionapproaches for determining the tissue level of the polypeptide of thepresent invention.

RNA molecules can be detected using methods known in the art includingfor example, Northern blot analysis, RT-PCR analyses, RNA in situhybridization stain and in situ RT-PCR stain.

Northern Blot analysis: This method involves the detection of aparticular RNA (e.g., the RNA molecule encoding LOXL2) in a mixture ofRNAs. An RNA sample is denatured by treatment with an agent (e.g.,formaldehyde) that prevents hydrogen bonding between base pairs,ensuring that all the RNA molecules have an unfolded, linearconformation. The individual RNA molecules are then separated accordingto size by gel electrophoresis and transferred to a nitrocellulose or anylon-based membrane to which the denatured RNAs adhere. The membrane isthen exposed to labeled DNA probes. Probes may be labeled usingradio-isotopes or enzyme linked nucleotides. Detection may be usingautoradiography, colorimetric reaction or chemiluminescence as describedhereinabove. This method allows both quantitation of an amount ofparticular RNA molecules and determination of its identity by a relativeposition on the membrane which is indicative of a migration distance inthe gel during electrophoresis.

RT-PCR analysis: This method uses PCR amplification of relatively rareRNAs molecules. First, RNA molecules from a particular tissue (e.g., acolon tumor tissue) are purified and converted into complementary DNA(cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) andprimers such as, oligo dT, random hexamers or gene specific primers, allof which are available from Invitrogen Life Technologies, Frederick,Md., USA. Then by applying gene specific primers and Taq DNA polymerase,a PCR amplification reaction is carried out in a PCR machine. Those ofskills in the art are capable of selecting the length and sequence ofthe gene specific primers and the PCR conditions (i.e., annealingtemperatures, number of cycles and the like) which are suitable fordetecting specific RNA molecules.

RNA in situ hybridization stain: In this method DNA or RNA probes areattached to the RNA molecules present in the tissue. Generally, a tissuesample (e.g., a colon tissue) is fixed to preserve its structure and toprevent the RNA from being degraded and then sectioned for microscopyand placed on a slide. Alternatively, frozen tissue samples can be firstsectioned and put on a slide and then subject to fixation prior tohybridization. Hybridization conditions include reagents such asformamide and salts (e.g., sodium chloride and sodium citrate) whichenable specific hybridization of the DNA or RNA probes with their targetmRNA molecules in situ while avoiding non-specific binding of probe.Those of skill in the art are capable of adjusting the hybridizationconditions (i.e., temperature, concentration of salts and formamide andthe like) to specific probes and types of cells. Followinghybridization, any unbound probe is washed off and the slide issubjected to either a photographic emulsion which reveals signalsgenerated using radio-labeled probes or to a colorimetric reaction whichreveals signals generated using enzyme-linked labeled probes asdescribed hereinabove.

In situ RT-PCR stain: This method is described in Nuovo et al., Am. J.Surg. Pathol. 17:683-690 (1993) and Komminoth et al., Pathol. Res.Pract. 190:1017-1025 (1994). Briefly, the RT-PCR reaction is performedon fixed tissue sections by incorporating labeled nucleotides to the PCRreaction. The reaction is carried on using a specific in situ RT-PCRapparatus such as the laser-capture microdissection PixCell I LCM systemavailable from Arcturus Engineering (Mountainview, Calif.).

Determination of an activity level of the polypeptide of the presentdisclosure (e.g., LOXL2) in a colon tumor tissue may be effected usingsuitable substrates in a cytochemical stain and/or in vitro activityassays.

Cytochemical stain: According to this method, a chromogenic substrate isapplied on the colon tumor tissue containing an active enzyme (e.g.,LOXL2). The enzyme catalyzes a reaction in which the substrate isdecomposed to produce a chromogenic product visible by a light or afluorescent microscope.

In vitro activity assays: In these methods the activity of a particularenzyme is measured in a protein mixture extracted from the tissue ofinterest (e.g., a colon tumor tissue). The activity can be measured in aspectrophotometer well using colorimetric methods (see for example,Wande et al., Proc. Natl. Acad Sci. USA. 1997, 94: 12817-12822 (1997))or can be measured in a non-denaturing acrylamide gel (i.e., activitygel). Following electrophoresis the gel is soaked in a solutioncontaining a substrate and colorimetric reagents. The resulting stainedband corresponds to the enzymatic activity of the polypeptide ofinterest (e.g., LOXL2). If well calibrated and within the linear rangeof response, the amount of enzyme present in the sample is proportionalto the amount of color produced. An enzyme standard is generallyemployed to improve quantitative accuracy.

Once the tissue level and/or the activity level of the polypeptide (ormRNA) of the present disclosure (e.g., LOXL2) is determined in the colontumor tissue the malignancy of the tumor is assessed by comparing theexpression level and/or activity in the colon tumor tissue to that of anormal colon tissue.

It will be appreciated that the normal colon tissue may be obtained froma biopsy and/or a surgery of a colon tissue obtained form a healthyindividual. Alternatively, the normal colon tissue can be obtained froman unaffected segment of the colon of the same individual. Methods ofdetermining the status of a normal colon tissue are known to skilled inthe art and include for example, a morphological evaluation of tissuesections.

Once malignancy of colon cancer is determined as described above, tissuelevel and/or activity level of the polypeptide (or mRNA thereof) of thepresent disclosure can also be utilized to stage the colon tumor and tothereby predict the prognosis of an individual diagnosed with coloncancer.

Such staging can be effected by assessing the tissue level and/oractivity level of the polypeptide and correlating it to results obtainedfrom colon cancer tissue at various stages (obtainable throughpathological evaluation of colon tumors). It will be appreciated thatsuch accurate and rapid staging will enable accurate and rapid prognosisof an individual afflicted with colon cancer and timely administrationof suitable treatment regimen.

Additional objects, advantages, and novel features of the presentdisclosure will become apparent to one of skill in the art. It isappreciated that certain features of the disclosure, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the disclosure, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. Additionally, each of the variousembodiments and aspects of the present disclosure as delineatedhereinabove and as claimed in the claims section below findsexperimental support in the following examples.

Examples

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present disclosure include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, (1989); Ausubel (Ed.),Current Protocols in Molecular Biology, Volumes I-III, John Wiley andSons, Baltimore, Md. (1994); Perbal, A Practical Guide to MolecularCloning, John Wiley & Sons, New York 0988); Watson et al., RecombinantDNA, Scientific American Books, New York; Birren et al. (Eds.), GenomeAnalysis: A Laboratory Manual Series, Vols. 1-4, Cold Spring HarborLaboratory Press, New York (1998); methodologies as set forth in U.S.Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;Cellis (Ed.), Cell Biology: A Laboratory Handbook, Volumes I-III,(1994); Coligan (Ed), Current Protocols in Immunology, Volumes I-III(1994); Stites et al. (Eds.), Basic and Clinical Immunology (8thEdition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi(Eds.), Selected Methods in Cellular Immunology, W. H. Freeman and Co.,New York (1980); available immunoassays are extensively described in thepatent and scientific literature, see, for example, U.S. Pat. Nos.3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517;3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074;4,098,876; 4,879,219; 5,011,771 and 5,281,521; Gait (Ed),Oligonucleotide Synthesis, (1984); Hames and Higgins (Eds.), NucleicAcid Hybridization, (1985); Hames and Higgins (Eds.), Transcription andTranslation, (1984); Freshney (Ed), Animal Cell Culture, (1986);Immobilized Cells and Enzymes, IRL Press (1986); and Methods inEnzymology, Vol. 1-317, Academic Press; PCR Protocols: A Guide ToMethods And Applications, Academic Press, San Diego, Calif. (1990);Marshak et al., Strategies for Protein Purification andCharacterization—A Laboratory Course Manual, CSHL Press (1996).

Example 1 ShRNA Against LOXL2 Inhibits Tumor Cell Invasiveness

Materials and Methods

Lentiviral expression plasmids containing several candidate DNAsequences encoding candidate shRNA species directed against LOXL2 and aDNA encoding a gene that confers resistance to the selective agentpuromycin were bought by us from Sigma (St. Louis, Mich.) (FIG. 1).Lentiviruses containing these candidate cDNAs were produced in theHEK293-T packaging cell line by transfection of the plasmids into thecells along with the packaging vector pCMVdR8.91, and a plasmid encodingthe vesicular stomatitis virus coat envelope pMD2-VSVG (5 μg).Recombinant replication defective lentiviruses were collected from theconditioned medium of the packaging cells and used to infect targettumor cells. The tumor invasion assay is illustrated in FIG. 2. Tumorcells are seeded between two layers of collagen in a monolayer, whichrepresents the tumor mass. The cells invade over time the adjacentlayers of collagen. The number of invading cells to various depths inmicroscopic fields is counted automatically using the Image-Promorphometric analysis software.

Results

The different lentiviruses carrying the different candidate DNA speciesencoding the different shRNAs were screened for their ability to inhibitthe expression of LOXL2. None of these cDNA species has any homology tothe sequences of other members of the lysyl-oxidase family of genes. Twoof these cDNA species were found to encode shRNA species that inhibitthe expression of LOXL2 at the mRNA and protein level. The DNA sequenceencoding the first shRNA is GAAGGAGACATCCAGAAGAAT (sh.LOXL2.197 orsi-197; SEQ ID NO: 20) and the second has the sequenceCGATTACTCCAACAACATCAT (sh.LOXL2.195, or si-195; SEQ ID NO: 21). Of thesetwo, si-195 is believed to be a slightly more potent inhibitor (FIG.3A).

The invasive/metastatic phenotype is associated in many instances with atransition from an epithelial to a mesenchymal morphology (EMT).Expression of the shRNA species in the human derived tumorigenic celllines induced a dramatic shift in morphology from a mesenchymal to anepithelial morphology (FIG. 4).

In order to assess the effects of the shRNA species on the invasivenessof the cells, tumor cells injected with control lentiviruses or tumorcells infected with the lentiviruses encoding the si-195 shRNA wereseeded between two layers of collagen and their ability to invade thecollagen above and below the monolayer of cells assessed. It can be seenthat expression of the si-195 shRNA substantially inhibited theinvasiveness of the cells into the collagen layers above and below theoriginal monolayer of cells (FIG. 5).

Example 2 LOXL2 is Overexpressed in Primary Alveolar Proteinosis

Primary alveolar proteinosis (PAP) is characterized by over-secretion oflung surfactant, and is a problematic condition of unknown etiology,which presents difficulties for both diagnosis and effective treatment.

Expression of LOXL2 and LOXL3 was assessed in lungs of normal and PAPpatients. FIG. 6 shows overexpression of LOXL2 in pulmonary endothelialcells in PAP, while LOXL3 is expressed in the smooth muscle.

Example 3 LOXL2 shRNA Promotes MET in Malignant Human Cells

LOXL2-induced EMT in three types of malignant human cells, 10HT1080,MDA-MB-231, or Yu/PAC2 cells. 2×10⁵ cells, were seeded in 35 mm dishes.The cells were infected with lentivirus directing expression of LOXL2shRNA, shRNA.Loxl2.195 or control shRNA (FIG. 7) according to the vendorprotocol (Sigma). Phalloidin staining of cells was performed by fixingcells in 4% paraformaldehyde. The cells were then permeabilised with0.05% saponin before being stained with rhodamine-phalloidin (MolecularProbes, Eugene, Oreg.) (FIG. 7). Cells treated with shRNA.Loxl2.195appeared to be in mesenchymal-epithelial transition (MET) states.

Calcium Phosphate Transfections:

Calcium phosphate transfections were performed for retroviralproduction. Generally, the plates were pre-coated with 0.2% sterilizedGelatin to ensure better adherence of the cells during all phases of theprotocol. For 100 mm plate ˜2.5-3.0×10⁶ cells were seeded for 293, 293Tcells and 3.2-3.5×10⁶ cells for ΦNX-A cells 18-24 hrs prior totransfection. For 150 min plates ˜6.0×10⁶ 293, 293T or ΦNX-A cells wereseeded.

Cells were ˜60% confluent at the time of transfection. For 100 mmplates, ˜10 μg DNA, ˜438 μl H2O (depending on volume of DNA), 61 μl 2MCaCl₂ (total volume of H₂0/DNA/CaCl₂ mixture: 500 μl) was mixed in amicrofuge tube. CaCl₂ was not added until the precipitates were ready tobe prepared. For 150 mm plates, 30 μg DNA, ˜878 μl H2O (depending onvolume of DNA), 122 μl 2M CaCl₂ (total volume of H₂0/DNA/CaCl₂ mixture:1000 μl) was used.

Lentiviral Production

For lentiviral production in 293 or 293T cells by Triple DNAco-transfection or to VSVg coat moloney viruses, using 100 mm plates,˜10 μg of three DNAs consisting of 5 μg of transfer vector, 3-4 μg ofstructural protein vector and 1-2 μg of pCI(VSVg) envelope vector wasused. For 150 mm plates, ˜30 μg of three DNAs (16 μg of lenti-transfervector, 12 μg of Δ8.2 structural vector and 3 μg of pCI(VSVg) envelopevector). If the transfer vector does not have a GFP gene a GFPexpression vector was usually included as ˜5% of the total DNA, to knowthat the relative efficiency of the transfection (at the time of viralharvesting).

500 μl of 2×HBS (50 mM Hepes, 10 mM KCl, 12 mM Dextrose, 280 mM NaCl,1.5 mM Na₂HPO₄×7H₂0) was added to the H₂0/DNA/CaCl₂ mixtures (100 mmplates) or 1000 μl (for150 mm plates) with constant bubbling of thelatter mixtures in either microcentrifuge tubes (if precipitates are 0.5ml of less) or 14 ml polypropylene tubes (if precipitates are 1-3 mleach) or into 15 ml polypropylene tubes. Then the CaCl₂ to the H₂0/DNAmixtures were added but to no more than four tubes at a time. Theprecipitates were then left at room temperature for 0-10 min.

Cells were in 9 ml of media (100 mm plates) or 18 ml media (150 mmplates). Excess media was removed leaving appropriate volumes in eachsize plate. Chloroquine was added to each plate to a final concentrationof 25 μM.

The 1 ml H₂0/DNA/CaCl₂/2×HBS precipitates were gently added to thecells. Microscopic examination should reveal very fine black particles.

Plates were put in a 37° C., 5% CO₂ incubator. To ensure that theprecipitates were evenly distributed plates were swirled gently by hand˜two times over the first 20-30 min of incubation.

5-8 hrs post-transfection the media was changed with 10 ml fresh GM(DMEM, 1% Penn/Strep, 1% glutamine, 10% FBS) for 100 mm plates and 22 mlfor 150 mm plates. 293 cells are more sensitive to chloroquine than ΦNXcells so the media was changed no longer than 5-7 hr post-transfection.

24 hrs post-transfection the GM was changed to 6.5-7 ml for the viralharvest (100 mm plates) or 16-17 ml (150 mm plates). 6 hours prior toharvesting plates were moved to a 32° C., 5% CO₂ incubator. Thesupernatants were centrifuged at 2000 RPM for 5 minutes to remove anycellular debris or were filtered thru a 0.45 micron filter.

Moloney and Lenti Retroviral Infections (Spinoculation Protocol)

To boost viral infection, adherent and suspension cells in tissueculture plate carriers can be spin infected at 2000-2400 RPM for 45-60minutes at 30-32° C. Cells were infected at a high MOI with undilutedvirus in 12, 24 or 6 well plates (generally using no more than50-100×10³ cells per well of a 6 well plate) in the presence of 8 μg/mlof polybrine. The cells were infected with 0.5-1.0 ml of viralsupernatant per well of a 24 well plate, 1 ml per well of a 12 wellplate and 2.0 ml/well of a 6 well plate. Viral supernatants from −80° C.freezer were thawed at room temperature and then added to cells alongwith the polybrine. For suspension cells, a counted number of cells wereresuspended directly in the undiluted viral supernatant. The plate wassealed with parafilm all around to avoid evaporation and pH changes inthe media during the spinoculation. After the first ‘Spinoculation’ theparafilm is removed from the plates and the viral supernatants arereplaced with a fresh aliquot of virus and polybrine (thawed virus canbe kept at room temperature during the first 45 minute spin) for asecond equivalent spin or even a third spin (depending on the toleranceof the cells to centrifugation). For most cells, cells were usually spuntwice (45 min each spin). For suspension cells, an additional equalvolume of fresh viral supernatant was added to each well for a secondspin. The parafilm was removed from the plates and without removing theviral supernatant, and continued to incubate at 32° C. in a CO₂incubator for up to a total of 4-6 hr from the time of the firstspinoculation (if three spins of 45 min. each was performed, theincubation with the last aliquot of virus was performed for another 2-3hrs at 32° C.). The regular growth media was changed and the cells wereincubated at 37° C. for 2 days prior to examining cells for GFPfluorescence. Drug selections were commenced 48 hrs post infection. Ifcells overgrew before reaching 48 hr, they were divided into more wellswith regular growth media.

Example 4 LOXL2 shRNA Decreases Primary Tumor Development

Five million MDA-MB-231 cells were infected with control shRNA encodinglentiviruses or with lentiviral vector expressing the 195 LOXL2 directedshRNA, shRNA.Loxl2.195 (si-LOXL2). Infected cells were selected prior tothe injection with puromycin (2 micrograms/nil). Two days beforeinjection, LOXL2 expression was determined in control vs shRNA.Loxl2.195infected cells and the inhibition was about 65% based upon densitometryof Western blot (direct reading of fluorescence from the blot). The rateof proliferation of control vs shRNA.Loxl2.195 infected cells in vitrowas similar. The infected cells were injected into the mammary fat padsof balb/c nu/nu female mice. Tumor volume was measured 6, 11, 14, 18,22, 25, and 27 days after injection. (FIG. 8A) Tumor development wasdecreased in mice injected with MDA-231 si-control compared to miceinjected with MDA-231 si-Loxl2 cells. (FIG. 8A, B)

Example 5 Genes in MCF7 Cells Affected by LOXL2 Overexpression orInhibition

MCF7 cells were transfected with expression vector to overexpress LOXL2(clone 12 and 14), infected with control lentiviral vector containing anon-related shRNA (WT), or infected with a mutant of LOXL2 that isenzymatically inactive due to a mutation in its LTQ motif (Y689F). Geneexpression was measured by RT-PCR and Western blotting. RT-PCR wasperformed using standard protocols. Increase in expression wasidentified in a number of genes when LOXL2 was overexpressed (FIG. 9A)

Cells were infected with lentiviral shRNA vector 195. Cells wereselected with puromycin and LOXL2 expression monitored by Western blot.RNA was isolated and RT-PCR was performed. PCR conditions using LOXL2specific primers was 30 cycles of 55, 72, 95° C., 1 min. each (FIG. 9B)

Example 6 LOXL2 Expression is Enhanced by Hypoxia

Cells were incubated in a hypoxia chamber at 37° C. at 1.5% O₂ for 24 h.A control was incubated in normoxic conditions (regular incubator). RNAwas prepared from the Δ549 cells and amplified by RT-PCR (30 cycles of55, 72, 95° C., 1 min. each). LOXL2 expression is increased underhypoxic conditions (FIG. 10A)

LOXL2 levels were assessed by Western blot. Cells were stimulated forthe indicated times with the indicated concentration of CoCl₂ (FIG. 10B)Equal concentrations of cell lysate was prepared with lysis buffercontaining 0.1% DOC and 1% NP40 with protease inhibitors and 10 mM Hepesbuffer, pH-7.2. Cell lysates were separated on an 8%-10% gradientSDS/PAGE gel, blotted and probed with anti-LOXL2 antibodies. Membraneswere stripped and reprobed with an antibody directed against β-actin toverify equal loading.

Example 7 Human Umbilical Vein Derived Endothelial Cells Contain CellSurface Bound LOXL2 Receptor

Four different cell lines, HUVEC, LE2, HMEC, and Balb, were tested fortheir specific binding to LOXL2 by measuring using iodinated LOXL2. toLOXL2 was iodinated and added to each well at a final concentration of0.35 μg/ml. Competition was done with unlabeled LOXL2 (3.5 μg/ml) (FIG.11A).

Wells were coated with gelatin, laminin or fibronectin to test for theirability to bind LOXLl2. LOXL2 was iodinated and added to each well at afinal concentration of 0.35 μg/ml. Competition was done with unlabeledLOXL2 (3.5 μg/ml). No specific binding was determined (FIG. 11B)indicating binding observed in FIG. 11A was not caused by binding tothese ECM components.

The same binding conditions as described in FIG. 11A above but in theabsence or addition of 100 ug/ml heparin or after prior digestion withheparinase. Neither absence nor addition of heparin of binding affectsLOXL2 binding. (FIG. 11C).

Example 8 LOXL2 and LOXL3 are Expressed in Neuronal Cells

Formalin fixed paraffin embedded 5 um sections from normal brain cortexwere examined by in situ hybridization. (FIG. 12)

Preparation of DIG-Labeled RNA probe

Digoxygenine (DIG-11-UTP)-labeled RNA probes were prepared in eithersense or antisense orientation. The probes were synthesized by run-offin vitro transcription using T7 RNA polymerase and the Dig/Genius RNAlabeling kit (Roche Boheringer-Manheim). Probes were generated by PCRreaction from human LOX, LOXL1, LOXL2, and LOXL3 cDNAs corresponding tonucleotides (starting from the first ATG) 164-560 for LOX, 668-1093 forLOXL1, 403-1102 for LOXL2, and 479-1913 for LOXL3. The primers werespecific for each probe and carried restriction enzymes for cloning tothe pBluescript (KS, SK) vector. The primers were:

hLOX: 5′ (EcoRI) CGCCGGAATTCGCTCACAGTACCAGCCTCAG 3′ (PstI)CCAAAACTGCAGGTAGTAGTTGTAATAAGGGT hLOXL1: 5′ (EcoRI)CGCCGGAATTCGGTCATCTACCCCTACCAGC 3′ (XbaI) CTAGTCTAGAACATAGTTGGGGTCTGGGAC

hLOXL2: LOXL2/pCDNA3.1 hygro was digested with EcoRA-NotI and fragmentwas cloned

hLOXL3: LOXL3/pCDNA3.1 hygro was digested with Xho-BglII and fragmentwas cloned.

The fragments were cloned into pBluescript in “KS” or “SK” orientationin order to generate “sense” or “antisense” probes.

The reaction mixture for problem labeling contained: 1 ug of DNA (LOXL2,LOX, LOXL1, or LOXL3 in pBluescript), 10× reaction buffer(Boehringer-Mannheim T7-RNA polymerase kit), 100 mM DTT (Sigma), 10×Dig-labeled dNTPs (Boehringer-Manneheim, Cat. No. 1175025), RNAseinhibitor (Promega, 15 units per reaction), 10× T7-RNA polymerase, andDEPC (diethyl pyrocarbonate)-treated water to complete volume of 20 ul.The reaction was for 2 hours at 37° C.

The reaction was stopped by addition of 0.8u1 EDTA. The Dig-labeled RNAprobe was then precipitated by the addition of 1 μl of 20 mg/mlglycogen, 2 ul 4M LiCl, and 55 ul chilled ethanol. The solution wasmixed well and incubated overnight at −70° C. The probe was pelleted bycentrifugation for 15 minutes at 4° C. at 13000 g. The precipitate waswashed with cold 70% ethanol and spun again for 5 minutes at 4° C. at13000 g. The pellet was dried and resuspended in 100 ul DEPC treated DDW(distilled deionized water). Four μl of probe was separated on regulated1% TAE agarose gel with 0.005% ethidium bromide to determine RNA probeformation. Final probe concentration for hybridization was 1 ug/ml.

Pre-Treatment of Slides

Formaline-fixed paraffin-embedded 5 um tissue sections weredeparaffinized by three washes of 5-10 min. each with 100% xylene. Thesections were rehydrated through a series of 100%, 95%, 70%, and 30%ethanol washes (5 min. incubation in each solution) at room temperature.The slides were then washed twice with DEPC treated water for 2 min. andthen treated with 0.2M HCl for 10 min to denature proteins. The sectionswere then washed twice with PBS and washed twice with water andsubsequently digested with Proteinase-K (20 ug/ml in TE, 10 min at roomtemperature). The reaction was stopped by two washes with DEPC treatedwater. The slides were then washed twice with PBS for 2 min.

Hybridization

Pre-hybridization was performed by incubation of slides for 2 hours at45° C. with preheated (at 70° C.) hybridization solution (see below).The probe was diluted in the preheated hybridization solution to a finalconcentration of 1 μg/ml and denatured by incubation at 80° C. for 5 minand was chilled for 3 min in ice prior to addition to the slides. Theslides were dried and the probes were added to the slides. The sectionswere covered with parafilm and incubated in a humidified chamberovernight at 45° C.

Post-Hybridization Washes and Incubation with Anti DIG Antibody

The slides were washed briefly with 2×SSPE at room temperature for 5min. and then incubated with 0.2×SSPE at 50-55° C. for 1 hour. Theslides were then washed by additional incubation with 0.2×SSPE at 50-55°C. for 1 hour and cooled to room temperature. After washing with PBS for5 min. at room temperature, the slides were incubated with Buffer 2 (seebelow) for 45 min. at room temperature with gentle agitation. The slideswere washed with BSA wash solution for 45 min. at room temperature withgentle shaking.

The anti-DIG antibody was diluted to final concentration of1:1000-1:1500 in Buffer 2 and added to the slides (˜150 ul/slide). Theslides were covered with parafilm and incubated in a humidified chamberovernight at 4° C.

The slides were then washed three times with BSA wash solution, for 5min. each wash. The slides were then washed with Buffer 2 for 30 min atroom temperature with gentle agitation.

Detection of Bound Anti-DIG Antibodies

The slides were incubated in Buffer 3 (see below) for 2 min. at roomtemperature without shaking. For colorimetric detection, 3 ul BCIP and4.2 ul NBT were diluted in 2 ml of Buffer 3. The solution was added tothe slides and incubated in the dark at room temperature for requiredtime. The reaction was stopped by addition of Stop Buffer (see below).The slides were then washed with DDW and counter stained with MayersHematoxylin (1:10 in DDW for 5-7 sec). Finally the slides were washedwith DDW and mounted with Mount Q and covered with coverslips.

Solutions

All solutions were prepared in DEPC treated water (0.1% DEPC in DDW for18 hours and then autoclaved).

Prehybridization Solution: 50% formamide, 100 mM Tris (pH 7.6), 150ug/ml tRNA, 1 mg/ml yeast total RNA, 10% Dextran sulfate, 300 mM NaCl, 1mM EDTA, 1% blocking reagant)

BSA Wash Solution: 1% BSA, 0.3% Triton X-100, 100 mM Tris (pH 7.6), 150mM NaCl.

Buffer 1: 100 mM Tris (pH 7.6), 150 mM NaCl

Buffer 2: blocking reagent diluted to 2% in Buffer 1

Buffer 3: 100 mM Tris (pH 9.5), 100 mM NaCl, 50 mM MgCl₂

Stop Buffer: 100 mM Tris (pH 8.0), 1 mM EDTA

“GVA-Mount solution” from Zymed (Cat. No. 00-8000)

Although the present disclosure has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

1. An isolated polynucleotide comprising a first sequence hybridizableto a polynucleotide sequence encoding SEQ ID NO: 2, a second sequencecomplementary to the first sequence, and a linking sequence that joinsthe first sequence to the second sequence.
 2. The isolatedpolynucleotide of claim 1, wherein the linking sequence forms a hairpinloop structure.
 3. The isolated polynucleotide of claim 1, wherein thefirst sequence comprises SEQ ID NO. 20 or
 21. 4. An isolatedpolynucleotide comprising SEQ ID NO.
 20. 5. The isolated polynucleotideof claim 4, wherein the isolated polynucleotide is at least twice thelength of SEQ ID NO.
 20. 6. The isolated polynucleotide of claim 4further comprising a sequence complementary to SEQ ID NO.
 20. 7. Theisolated polynucleotide of claim 4, wherein the polynucleotide comprisesa hairpin loop structure.
 8. An isolated polynucleotide comprising SEQID NO.
 21. 9. The isolated polynucleotide of claim 8, wherein theisolated polynucleotide is at least twice the length of SEQ ID NO. 20.10. The isolated polynucleotide of claim 8 further comprising a sequencecomplementary to SEQ ID NO.
 21. 11. The isolated polynucleotide of claim8, wherein the polynucleotide comprises a hairpin loop structure.
 12. Anexpression vector comprising the polynucleotide of claim
 1. 13. A hostcell comprising the expression vector of claim
 12. 14. A pharmaceuticalcomposition comprising: (a) a polynucleotide comprising a first sequencehybridizable to a polynucleotide sequence encoding SEQ ID NO. 2, asecond sequence complementary to said first sequence, and a linkingsequence that joins said first sequence to said second sequence; and,(b) a pharmaceutical excipient.
 15. A pharmaceutical compositioncomprising: (a) a polynucleotide comprising SEQ ID NO. 20 or 21; and,(b) a pharmaceutical excipient.
 16. A method for inhibiting apathological process in a subject, wherein the method comprisesadministering to the subject an effective amount of a polynucleotide,wherein the polynucleotide comprises a first sequence hybridizable to apolynucleotide sequence encoding SEQ ID NO. 2, a second sequencecomplementary to the first sequence, and a linking sequence that joinsthe first sequence to the second sequence; and wherein the pathologicalprocess is selected from the group consisting of tumor growth,metastasis, fibrosis and angiogenesis. 17.-19. (canceled)
 20. The methodof claim 16, wherein the linking sequence forms a hairpin loopstructure.
 21. The method of claim 16, wherein the first sequencecomprises SEQ ID NO. 20 or
 21. 22. A method for inhibiting apathological process in a subject, wherein the method comprisesadministering to the subject an effective amount of a polynucleotide,wherein the polynucleotide comprises SEQ ID NO. 20 or 21, and whereinthe pathological process is selected from the group consisting of tumorgrowth, metastasis, fibrosis, and angiogenesis. 23.-25. (canceled) 26.The method of claim 22, wherein the polynucleotide is at least twice thelength of SEQ ID NO.
 20. 27. The method of claim 22, wherein thepolynucleotide further comprises a sequence complementary to SEQ ID NO.20.
 28. The method of claim 22, wherein the polynucleotide comprises SEQID NO.
 21. 29.-31. (canceled)
 32. The method of claim 28, wherein thepolynucleotide is at least twice the length of SEQ ID NO.
 21. 33. Themethod of claim 28, wherein the polynucleotide further comprises asequence complementary to SEQ ID NO.
 21. 34. The method of claim 22,wherein the polynucleotide comprises a hairpin loop structure.
 35. Amethod for treating pulmonary alveolar proteinosis (PAP) in a subject,wherein the method comprises administering an effective amount of anagent to inhibit PAP in the subject, wherein the agent modulates theexpression or activity of a lysyl oxidase or lysyl oxidase like protein.36. A method for detecting pulmonary alveolar proteinosis (PAP) in asubject, wherein the method comprises administering to the subject anagent that detects an expression or activity of a lysyl oxidase or lysyloxidase like protein, wherein the expression or activity is used todiagnose PAP in the subject.
 37. The method of claim 35, wherein thelysyl oxidase like protein is LOXL2 or LOXL3.
 38. The method of claim35, wherein the agent is an antibody, a small molecule, antisensemolecule, ribozyme, DNAzyme, triple helix forming oligonucleotides,siRNA, or shRNA.
 39. The method of claim 35, wherein the agent is aninhibitor of the lysyl oxidase or lysyl oxidase like protein.